High intensity labeled reactant compositions and methods for sequencing

ABSTRACT

Compositions useful for the detection of single molecules in a sample are provided. In some aspects, the disclosure provides a nucleic acid connected to a nucleotide and two or more luminescent labels. In some embodiments, the nucleic acids described herein comprise one or more structural features that provide enhanced fluorescence intensity. In some aspects, methods of sequencing using the labeled nucleotides of the disclosure are provided.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/536,426, filed Jul. 24, 2017,which is hereby incorporated by reference in its entirety.

FIELD OF THE APPLICATION

The present application is directed generally to brightly labeledreactant compositions and methods of using the same for the detection ofsingle molecules.

BACKGROUND

Advancements in next-generation sequencing technologies have made itpossible to conduct massively parallel analysis of single molecules,which has fundamentally altered the landscape of life science research.Some of these techniques involve monitoring a biological reaction inreal-time using luminescently labeled reaction components. The labelsare illuminated with a light source to cause luminescence, and theluminescent light is detected with a photodetector. These events can berecorded and analyzed to identify individual reaction components basedon corresponding luminescent properties. In identifying a specific typeof labeled molecule among a plurality of types, it is critical that eachtype possess unique and readily identifiable luminescent properties.Furthermore, these parameters can be determinative of instrumentalrequirements such as excitation source power and overall instrumentsize.

SUMMARY

Aspects of the technology disclosed herein relate to labeled reactioncomponents comprising two or more luminescent labels separated by alinker (e.g., a constrained linker). In some embodiments, theapplication relates to the separation of luminescent labels to preventattenuation of detectable signals due to label-label interaction. Insome aspects, the application provides labeled nucleotides comprising anucleotide (e.g., a nucleoside polyphosphate) connected to two or moreluminescent labels via a linker. In some aspects, the applicationprovides compositions, methods, and kits for sequencing a templatenucleic acid.

In some aspects, the application provides labeled nucleotides comprisinga nucleotide (e.g., a nucleoside polyphosphate) connected to two or moreluminescent labels via a linker. In some embodiments, the nucleotide isconfigured for use as a substrate in a polymerization reaction. In someembodiments, labeled nucleotides of the application comprise two or moreluminescent labels separated from one another by a minimum distance. Insome embodiments, each luminescent label is at least 5 angstromsseparated from any other luminescent label. For example, in someembodiments, each luminescent label is at least 5, at least 10, at least15, at least 20, at least 25, at least 30, at least 40, or at least 50angstroms separated from any other luminescent label. In someembodiments, each luminescent label comprises a center of mass that isat least 5 angstroms separated from the center of mass of any otherluminescent label.

In some embodiments, labeled nucleotides of the application comprise oneor more luminescent labels attached to the linker via a spacer molecule.In some embodiments, the spacer molecule connects a luminescent label toan attachment site on the linker. In some embodiments, a luminescentlabel is attached to the linker via a spacer molecule that comprises atleast 8 contiguous atoms between the luminescent label and theattachment site on the linker. In some embodiments, the spacer moleculecomprises fewer than 50, fewer than 40, fewer than 30, or fewer than 20contiguous atoms between the luminescent label and the attachment siteon the linker. In some embodiments, a luminescent label is integratedinto the linker.

In some embodiments, the linker is an oligomer (e.g., an oligomericlinker, or a polymeric linker). In some embodiments, the oligomercomprises monomeric units. In some embodiments, the oligomer comprisestwo or more different types of monomeric units. In some embodiments, theoligomer comprises a plurality of the same type of monomeric unit (e.g.,the oligomer is a polymer of one type of monomeric units). In someembodiments, the oligomer comprises a first region with a plurality of afirst type of monomeric units and a second region with a plurality of asecond type of monomeric units. In some embodiments, the oligomercomprises a plurality of different regions (e.g., 2, 3, 4, 5, or more)each comprising a plurality of a different type of monomeric units. Insome embodiments, the oligomer comprises at least 5 monomeric units. Insome embodiments, the oligomer comprises at least 10 monomeric units. Insome embodiments, the oligomer comprises fewer than 150, fewer than 100,or fewer than 50 monomeric units (e.g., at least 5 monomeric units andfewer than 200, 150, 100, 75, 50, or 25 monomeric units; at least 10monomeric units and fewer than 200, 150, 100, 75, 50, or 25 monomericunits).

In some embodiments, where the linker is an oligomer (e.g., anoligomeric linker, a polymeric linker), each luminescent label isseparated from each other label by at least 5 monomeric units of theoligomer. In some embodiments, a first luminescent label is integratedinto the linker at a first position that is at least 5 monomeric unitsseparated from a second position at which a second luminescent label isintegrated into or attached to the linker. In some embodiments, whereadjacent luminescent labels are integrated into the linker, the labelscan be separated by fewer than 5 monomeric units. In some embodiments,each luminescent label is attached to the linker at an attachment sitethat is at least 5 monomeric units (e.g., at least 6, at least 8, atleast 10, at least 12, at least 14, at least 16, at least 18, at least20, or more, monomeric units) separated from any other attachment site.In some embodiments, each luminescent label is attached at an attachmentsite that is at least 5 monomeric units and fewer than 40 monomericunits (e.g., fewer than 38, fewer than 36, fewer than 34, fewer than 32,fewer than 30, fewer than 28, fewer than 26, fewer than 24, fewer than22, or fewer than 20 monomeric units) separated from any otherattachment site. In some embodiments, a luminescent label is integratedinto the linker in between two sequential monomeric units of theoligomer (e.g., covalently connecting two adjacent monomeric units of anoligomer).

In some embodiments, a linker is sufficiently rigid to preventinteractions between two or more labels connected to the linker. In someembodiments, the rigidity of the linker is sufficient to preserve atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, forexample 95-100% (e.g., around 95%, around 96%, around 97%, around 98%,around 99%, around 100%) of the intensity of each label relative totheir intensity when present as a single label attached to the samelinker.

In some embodiments, the linker is a peptide. In some embodiments, theamino acid composition of the peptide provides structural rigidity(e.g., due to the presence of one or more polyproline segments in thepeptide linker). In some embodiments, 90% or more (e.g., all) of thepeptide linker consists of a polyproline polymer. In some embodiments,the peptide rigidity is provided by constraining the peptide viachemical modification. For example, in some embodiments, the peptidecomprises one or more cyclized segments. In some embodiments, thepeptide is a cyclized peptide (e.g., a stapled peptide, an end-to-endcyclized peptide, etc.). In some embodiments, sufficient peptiderigidity can be provided by incorporating a combination of one or morerigid amino acid polymer segments and one or more chemically modifiedamino acid polymer segments.

In some embodiments, the linker is a polysaccharide (e.g., heparin,heparin sulfate, polyglucose, polylactose, aminoglycosides,N-acetylaminoglycosides, and combinations thereof).

In some embodiments, the linker is a nucleic acid. In some embodiments,the nucleic acid comprises a deoxyribonucleic acid (DNA), a ribonucleicacid (RNA), a peptide nucleic acid (PNA), a locked nucleic acid (LNA),or a derivative thereof. In some embodiments, sufficient rigidity isprovided by using one or more double stranded nucleic acid segments(e.g., separating two or more different labels). In some embodiments,sufficient rigidity is provided by one or more chemical modifications ofa nucleic acid (e.g., of a single-stranded or a double-stranded nucleicacid, or of a nucleic acid comprising one or more single-stranded andone or more double-stranded segments). In some embodiments, a nucleicacid comprises a combination of one or more double-stranded segments andone or more chemically modified segments.

In some embodiments, the nucleic acid comprises a combination of one ormore single-stranded and one or more double-stranded segments. Asingle-stranded segment can, in some embodiments, be present in the formof a loop (e.g., as in a stem-loop secondary structure describedelsewhere herein). In some embodiments, a single-stranded segment ispresent in the form of an unpaired region within a double-strandedsegment. For example, an internal loop can form within a double-strandedsegment where one or more bases of one strand do not form base pairinginteractions with one or more adjacent bases of the other strand. Afurther example of an unpaired region includes bulge loops, which canform within double-stranded segments where one strand includes one ormore additional bases relative to the other strand. In some embodiments,single-stranded regions and double-stranded regions impart structuralrigidity.

In some embodiments, a single-stranded region (e.g., an unpaired region)is at least 2 bases long (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more,bases long). In some embodiments, a single-stranded region is between 2and 10 bases long (e.g., between 2 and 8, between 4 and 10, or between 4and 8, bases long). In some embodiments, a double-stranded region isbetween 2 and 40 bases long (e.g., between 2 and 20, between 2 and 10,between 10 and 40, between 10 and 30, between 10 and 20, between 20 and40, or between 20 and 30, bases long).

In some embodiments, the nucleic acid comprises a first oligonucleotidestrand attached to the two or more luminescent labels. In someembodiments, the two or more luminescent labels are attached at two ormore attachment sites on the first oligonucleotide strand. In someembodiments, each luminescent label comprises a steric volume having acenter point that is at least 5 angstroms separated from that of anyother luminescent label. For example, in some embodiments, eachluminescent label comprises a steric volume having a center point thatis at least 6 angstroms, between about 5 to 10 angstroms, between about6 to 10 angstroms, between about 10 to 15 angstroms, between about 15 to20 angstroms, between about 20 to 25 angstroms, or between about 25 to50 angstroms separated from that of any other luminescent label.

In some embodiments, the nucleic acid further comprises a secondoligonucleotide strand hybridized with the first oligonucleotide strand.In some embodiments, the first oligonucleotide strand is attached to anucleotide (e.g., a nucleoside polyphosphate). In some embodiments, thesecond oligonucleotide strand is attached to a nucleotide (e.g., anucleoside polyphosphate).

In some embodiments, the two or more attachment sites are separated fromone another by at least 5 bases (e.g., at least 5 nucleotides) on thefirst oligonucleotide strand. In some embodiments, the two or moreattachment sites are separated from one another by at least 5 and fewerthan 40 bases (e.g., between about 5 and 30 bases, between about 5 and20 bases, between about 5 and 10 bases, between about 10 and 40 bases,between about 20 and 40 bases, or between about 30 and 40 bases) on thefirst oligonucleotide strand. In some embodiments, each attachment siteis at least 2 bases separated from a guanine or a cytosine on the firstoligonucleotide strand. In some embodiments, each attachment site occursat an abasic site on the first oligonucleotide strand. In someembodiments, each attachment site occurs at a nucleobase of a nucleotideon the first oligonucleotide strand. In some embodiments, the nucleobaseis selected from an A, T, or U nucleobase.

In some embodiments, the first oligonucleotide strand forms one or more(e.g., 1, 2, 3, 4, or more) stem-loops. In some embodiments, a loopregion of each stem-loop comprises an attachment site of the two or moreattachment sites. In some embodiments, the loop region of each stem-loopcomprises at least 4 unpaired bases (e.g., 4, 5, 6, 7, 8, or more,unpaired bases). In some embodiments, the loop region comprises asequence (e.g., a nucleotide sequence) having less than 33% G/C content.

In some aspects, labeled nucleotides of the disclosure comprise anucleic acid linker comprising a first oligonucleotide strand attachedto two or more branching oligonucleotide strands at a terminal end ofthe first oligonucleotide strand. In some embodiments, the firstoligonucleotide strand is attached to the two or more branchingoligonucleotide strands via a covalent coupling compound. In someembodiments, each branching oligonucleotide strand comprises at leastone luminescent label. In some embodiments, the first oligonucleotidestrand is hybridized with a second oligonucleotide strand. In someembodiments, the second oligonucleotide strand is attached to anucleotide (e.g., a nucleoside polyphosphate). In some embodiments, eachbranching oligonucleotide strand is further hybridized with acomplementary branching oligonucleotide strand.

In some embodiments, the covalent coupling compound is of a structure:

wherein N_(f) is the first oligonucleotide strand; N_(b) is a branchingoligonucleotide strand; R_(f) and R_(b) are each, independent from oneanother, a bond or a linking group selected from the group consisting ofsubstituted or unsubstituted alkylene; substituted or unsubstitutedalkenylene; substituted or unsubstituted alkynylene; substituted orunsubstituted heteroalkylene; substituted or unsubstitutedheteroalkenylene; substituted or unsubstituted heteroalkynylene;substituted or unsubstituted heterocyclylene; substituted orunsubstituted carbocyclylene; substituted or unsubstituted arylene;substituted or unsubstituted heteroarylene; and combinations thereof;and each instance of 0 is an oxygen atom of either a 5′ phosphate groupor a 3′ hydroxyl group of an adjacent oligonucleotide strand.

In some aspects, labeled nucleotides of the disclosure comprise anucleic acid linker comprising a first oligonucleotide component thatcomprises three or more oligonucleotide strands (e.g., 3, 4, 5, 6, ormore, oligonucleotide strands) extending from a covalent couplingcompound. In some embodiments, at least one of the three or moreoligonucleotide strands is attached to a nucleotide (e.g., a nucleosidepolyphosphate). In some embodiments, the first oligonucleotide componentis hybridized with a second oligonucleotide component. In someembodiments, the second oligonucleotide component comprises at least oneoligonucleotide strand attached to a luminescent label.

In some embodiments, labeled nucleotides of the disclosure areluminescently labeled with a fluorescent dye. In some embodiments, thefluorescent dye is a rhodamine dye, a BODIPY dye, or a cyanine dye.

In some embodiments, labeled nucleotides of the disclosure comprise anucleotide (e.g., a nucleoside polyphosphate) that is at least 1 nmseparated from any luminescent label of the two or more luminescentlabels. In some embodiments, the nucleotide is separated from anyluminescent label of the two or more luminescent labels by betweenapproximately 1 and 10 nm (e.g., between approximately 2 and 10 nm,between approximately 4 and 10 nm, between approximately 6 and 10 nm, orbetween approximately 8 and 10 nm). In some embodiments, the nucleotideis separated from any luminescent label of the two or more luminescentlabels by between approximately 2 and 20 nm (e.g., between approximately6 and 20 nm, between approximately 10 and 20 nm, between approximately12 and 20 nm, or between approximately 16 and 20 nm).

In some aspects, the disclosure provides methods of determining thesequence of a template nucleic acid. In some embodiments, the methodsinclude a step comprising exposing a complex in a target volume, thecomplex comprising the template nucleic acid, a primer, and apolymerizing enzyme, to a plurality of types of luminescently labelednucleotides provided by the application. In some embodiments, one ormore of the plurality of types of luminescently labeled nucleotidescomprise a nucleotide (e.g., a nucleoside polyphosphate) connected totwo or more luminescent labels via a linker. In some embodiments, thelinker is an oligomer that comprises at least 10 monomeric units. Insome embodiments, each luminescent label is attached to the linker at anattachment site that is at least 5 monomeric units separated from anyother attachment site. For example, in some embodiments, an attachmentsite is separated from any other attachment site by between about 5 and30 monomeric units, between about 5 and 20 monomeric units, betweenabout 5 and 10 monomeric units, between about 10 and 40 monomeric units,between about 20 and 40 monomeric units, or between about 30 and 40monomeric units. In some embodiments, each luminescent label is at least5 angstroms separated from any other luminescent label. For example, insome embodiments, each luminescent label is separated from any otherluminescent label by approximately 5 to 10 angstroms, approximately 6 to10 angstroms, approximately 10 to 15 angstroms, approximately 15 to 20angstroms, approximately 20 to 25 angstroms, or approximately 25 to 50angstroms. Accordingly, in some aspects, the disclosure provides methodsof nucleic acid sequencing that utilize any of the luminescently labelednucleotides described herein.

In some embodiments, the methods further comprise a step of directing aseries of pulses of one or more excitation energies towards a vicinityof the target volume. In some embodiments, the methods further comprisea step of detecting a plurality of emitted photons from luminescentlylabeled nucleotides during sequential incorporation into a nucleic acidcomprising the primer. In some embodiments, the methods further comprisea step of identifying the sequence of incorporated nucleotides bydetermining timing and optionally luminescent intensity and/orbrightness of the emitted photons.

In some embodiments, four different types of nucleotides (e.g., adenine,guanine, cytosine, thymine/uracil) in a reaction mixture can each belabeled with one or more luminescent molecules (e.g., having two or moreluminescent labels, as described herein). In some embodiments, each typeof nucleotide can be connected to more than one of the same luminescentmolecule (e.g., two or more of the same fluorescent dye connected to anucleotide). In some embodiments, each luminescent molecule can beconnected to more than one nucleotide (e.g., two or more of the samenucleotide). In some embodiments, more than one nucleotide can beconnected (e.g., via a linker described herein) to more than oneluminescent molecule.

In some embodiments, the luminescent labels among a set of fournucleotides can be selected from dyes comprising an aromatic orheteroaromatic compound and can be a pyrene, anthracene, naphthalene,acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole,benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline,ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluoroscein, rhodamine, or other like compound. Examples ofdyes include xanthene dyes, such as fluorescein or rhodamine dyes,naphthalene dyes, coumarin dyes, acridine dyes, cyanine dyes,benzoxazole dyes, stilbene dyes, pyrene dyes, phthalocyanine dyes,phycobiliprotein dyes, squaraine dyes, BODIPY dyes, and the like.

In some aspects, the application provides kits for sequencing a templatenucleic acid. In some embodiments, a kit comprises a plurality of typesof luminescently labeled nucleotides as described herein. In someembodiments, each type of labeled nucleotide comprises two or moreluminescent labels attached to one or more nucleotides (e.g., one ormore nucleoside polyphosphates) via a linker. In some embodiments, thekit further comprises a polymerizing enzyme. In some embodiments, thekit further comprises a primer complementary to the template nucleicacid being sequenced.

In some aspects, the application provides nucleic acid sequencingreaction compositions. In some embodiments, the compositions comprisetwo or more (e.g., 2, 3, 4, or more) different types of luminescentlylabeled nucleotides in a reaction mixture. In some embodiments, eachtype of luminescently labeled nucleotide comprises a labeled nucleotideaccording to the present application. In some embodiments, thecompositions further comprise a polymerizing enzyme. In someembodiments, the compositions further comprise a template nucleic acid.In some embodiments, the compositions further comprise a primercomplementary to the template nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that, in someinstances, various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings.

When describing embodiments in reference to the drawings, directionreferences (“above,” “below,” “top,” “bottom,” “left,” “right,”“horizontal,” “vertical,” etc.) may be used. Such references areintended merely as an aid to the reader viewing the drawings in a normalorientation. These directional references are not intended to describe apreferred or only orientation of an embodied device. A device may beembodied in other orientations.

As is apparent from the detailed description, the examples depicted inthe figures (e.g., FIGS. 1-10 ) and further described for the purpose ofillustration throughout the application describe non-limitingembodiments, and in some cases may simplify certain processes or omitfeatures or steps for the purpose of clearer illustration.

FIG. 1A is a diagram that generically depicts luminescent labelseparation on a brightly labeled reactant.

FIG. 1B depicts generic linker structures having different luminescentlabel attachment configurations.

FIG. 2A is a diagram that generically depicts a nucleic acid linkerattached to two luminescent labels.

FIG. 2B depicts generic nucleic acids having differing strandconfigurations (top) and a diagram that generically depicts luminescentlabel spatial occupation (bottom).

FIG. 2C depicts generic nucleic acids having different relative labelattachment sites (top) and different reactant connectivity (bottom).

FIG. 3A depicts generic nucleic acids connecting labels to a reactantvia same (left) or opposite (right) oligonucleotide strand connectivity.

FIG. 3B depicts generic nucleic acids with examples of approximate sizeconstraints that may be used in the design of brightly labeledreactants.

FIG. 3C is an example structure of a nucleic acid connecting twoluminescent labels to a nucleoside polyphosphate via same-strandconnectivity.

FIG. 3D depicts an example of a sequencing reaction which confirmed thata rod-shaped nucleic acid linker (e.g., as shown in FIG. 3C) can be usedto detect incorporation of a labeled nucleoside polyphosphate.

FIG. 3E is an example structure of a nucleic acid connecting twoluminescent labels to a nucleoside polyphosphate via opposite-strandconnectivity.

FIG. 3F is an example structure of a nucleic acid linker havingluminescent labels integrated into an oligonucleotide strand.

FIG. 3G depicts an example of a sequencing reaction that was conductedusing the four different nucleic acid linker constructs shown.

FIG. 4A depicts generic nucleic acid linkers having a single stem-loopsecondary structure.

FIG. 4B depicts generic nucleic acid linkers having multiple stem-loopsecondary structures.

FIG. 4C is an example structure of a nucleic acid linker havingluminescent labels attached at loops of separate stem-loop secondarystructures.

FIG. 4D depicts an example of a sequencing reaction which confirmed thata labeled nucleoside polyphosphate having a stem-loop nucleic acidlinker (e.g., as shown in FIG. 4C) can be used to detect incorporationof a nucleoside polyphosphate.

FIG. 5A generically depicts tree-shaped nucleic acid linkers havingbranching oligonucleotide strands attached to luminescent labels.

FIG. 5B is an example of a tree-shaped nucleic acid linker havingluminescent labels attached at terminal ends of separate branchingoligonucleotide strands.

FIG. 6A generically depicts star-shaped nucleic acid linkers andprovides an example of approximate size constraints that can be used inthe design of star-shaped nucleic acid linkers.

FIG. 6B is an example structure of a star-shaped nucleic acid linker.

FIG. 6C is an example structure of a nucleic acid linker having atetrahedral core.

FIG. 6D depicts an example of a reaction scheme that can be used tosynthesize a nucleic acid linker having a tetrahedral core.

FIG. 6E generically depicts a tetrahedral-based nucleic acid linkerstructure having luminescent labels attached at three-way junctions.

FIG. 7 generically depicts a process of generating a cyclodextrin-basednucleic acid linker having luminescent labels attached at three-wayjunctions.

FIG. 8 depicts examples of nucleic acid linker structures used in a setof experiments to evaluate the effects of spacer length on fluorescencelifetime measurements.

FIG. 9 depicts examples of nucleic acid linker structures used in a setof experiments to evaluate the effects of linker constraint onfluorescence lifetime measurements.

FIG. 10 depicts examples of nucleic acid linker structures used in a setof experiments to evaluate the effects of spacer length on fluorescencelifetime in constrained linkers.

DETAILED DESCRIPTION

Among other aspects, the disclosure provides luminescently labeledreactants comprising two or more labels, wherein the labels areconfigured to provide high intensity and/or consistent emissioncharacteristics (e.g., consistent emission lifetimes). In someembodiments, the two or more labels are configured to avoid label-labelinteractions that could reduce the intensity or other emissioncharacteristics of the emissions. In some embodiments, the two or morelabels are configured to a) avoid interactions with a linker, and/or b)to each have similar interactions with a linker.

In some aspects, the disclosure provides methods and compositionsrelated to luminescently labeled reactants having high emissionintensity. In some aspects, the disclosure provides methods andcompositions related to luminescently labeled reactants having highemission brightness. In some aspects, the disclosure relates to brightlylabeled reactants having consistent emission lifetime. As used herein,in some embodiments, “brightness” (and variations thereof, e.g.,“bright,” “brightly,” etc.) refers to a parameter that reports on theaverage emission intensity per labeled reactant molecule. Thus, in someembodiments, “emission intensity” may be used to generally refer tobrightness of a composition comprising brightly labeled reactants. Insome embodiments, brightness of a labeled reactant is equal to theproduct of its quantum yield and extinction coefficient. In someembodiments, the labeled reactants of the disclosure are engineered tomaximize quantum yield and/or minimize extinction coefficient values topromote increased brightness.

The brightly labeled reactants of the disclosure are, in someembodiments, engineered to have increased emission brightness andconsistent emission lifetime. In some embodiments, two labels of areactant can interact with one another and/or a surrounding environmentsuch that one or more emission characteristics are inconsistent betweenthe two labels. Inconsistent emission characteristics can beproblematic, in some embodiments, where single molecule detectionmethods rely upon these characteristics to identify a certain type ofmolecule. For example, in some embodiments, inconsistent emissionlifetime can result in lifetime readouts that report two separateclusters of information as opposed to a single grouping of informationthat would be observed with consistent emission lifetime.

In some embodiments, consistent emission lifetime can involve preservingemission lifetime, e.g., having approximately unchanged emissionlifetime relative to a labeled reactant having one fewer label. In someembodiments, emission lifetime of labels in a multiply-labeled reactantis unchanged relative to the same label in a singly-labeled reactant. Asdescribed herein, increasing the number of luminescent labels on asingle construct to increase brightness can, in some embodiments, resultin diminished emission lifetime. In some embodiments, the disclosureprovides compositions engineered using specific structural constraintsto separate adjacent labels by a certain minimum distance that increasesbrightness without affecting emission lifetime. In some embodiments,emission lifetime of a multiply-labeled construct is compared to theemission lifetime of a construct having at least one fewer luminescentlabel (e.g., at least one fewer of the same type of fluorophore dye). Insome embodiments, emission lifetime is altered by approximately 30% orless (e.g., increased or decreased by less than 30%, less than 25%, lessthan 20%, less than 15%, less than 10%, less than 5%, less than 4%, lessthan 3%, less than 2%, less than 1%, or approximately 0%).

In some aspects, the disclosure relates to the recognition andappreciation that the detectable compositions in a sequencing reactioncan be developed to eliminate the need for certain instrumentcomponents, thereby moving the technology toward more compact systems.For example, sequencing instruments generally require optical filters tofilter the excitation light from causing undesirable detection events atthe sensor. Optical filters used to transmit the desired luminescenceand sufficiently block the excitation light can be thick, bulky,expensive, and intolerant to variations in the incidence angle of light,preventing miniaturization. The inventors, however, recognized andappreciated that using brightly labeled reactants having preservedlifetimes can reduce the need for such filtering or, in some cases,remove the need for such filters altogether. The bright reactantsdescribed herein allow less optical power (e.g., excitation energy) tobe used, which reduces scattering and the requirement for filtering as aresult.

Aspects of the application provide brightly labeled reactants configuredaccording to the non-limiting diagram shown in FIG. 1A, which depictstwo luminescent labels connected to a reactant 180 via a linker 100. Asshown, each luminescent label is attached to the linker 100 via aspacer. The spacer, in some embodiments, forms a covalent bridge betweenlabel and linker. As such, in some embodiments, the spacer is neitherpart of the luminescent molecule nor is the spacer part of the linkercomposition (e.g., the spacer does not contain a monomeric unit of apolymeric or oligomeric linker). A first end of the spacer attaches to alinker attachment site 102, and a second end of the spacer attaches to alabel attachment site 122. In some embodiments, the linker attachmentsite 102 can be approximated by the location of the covalent bondjoining an atom of the spacer to an atom of the linker 100. In someembodiments, the linker attachment site 102 occurs at an atom that iswithin a contiguous chain of the linker 100. In some embodiments, thelabel attachment site 122 can be approximated by the location of thecovalent bond joining an atom of the spacer to an atom of the label. Insome embodiments, the label attachment site 122 occurs at the atom ofthe label that is covalently bound to the atom of the spacer. In someembodiments, the label attachment site 122 occurs at the atom of thespacer that is covalently bound to the atom of the label.

As described elsewhere herein, in some embodiments, linker constructs ofthe application comprise two or more luminescent labels, where adjacentluminescent labels are described as having attachment sites separated bya minimum distance d_(A). In some embodiments, each luminescent label isseparated from the next by a minimum distance d_(L). As shown in FIG.1A, in some embodiments, d_(L) is the distance between label attachmentsites. In some embodiments, label-label separation can be furtherdictated by the size of each label molecule. Accordingly, in someembodiments, luminescent labels can be described by an approximated orcalculated steric volume 112 of an ellipsoid or a spheroid to obtain ameasurement for the steric radius, r₁. In some embodiments, luminescentlabels can be described by an approximated or calculated stericcircumference of an ellipse or a circle to obtain a measurement for thesteric radius, r₁. In some embodiments, steric radius (e.g., r₁, r₂) canbe calculated or approximated as one-half of the longest dimension of aluminescent label. For example, in some embodiments, the chemicalstructure of a luminescent label is evaluated as a two-dimensional orthree-dimensional structure (e.g., based on athermodynamically-favorable molecular conformation) using software or asuitable method known in the art, and a steric radius (e.g., r₁, r₂) isdetermined by calculating one-half of the structure's longest dimensionin two-dimensional or three-dimensional space. In some embodiments,labels are separated by minimum distance d_(L), provided that theaggregate label radii (r₁+r₂) are such that the labels do not overlap.

In some embodiments, as illustrated in FIG. 1B, linker configurationand/or spacer rigidity is such that distance between attachment sitesd_(A) can be approximately the same as d_(L) (e.g., as in construct150). In some embodiments, linker configuration and/or spacer rigidityis such that distance between attachment sites d_(A) can be less thanminimum distance d_(L) (e.g., as in construct 152). In some embodiments,linker configuration and/or spacer rigidity is such that distancebetween attachment sites d_(A) can be greater than minimum distanced_(L) (e.g., as in construct 154).

It should be appreciated that, in some embodiments, the conceptsdescribed herein can be implemented using any suitable molecularscaffold as a linker 100. In some embodiments, the linker is an organiccompound. Examples of organic compounds suitable for use as a linker 100include, without limitation, polyphenyls, polyalkynes, alpha helixmimetics, and peptidomimetics.

In some embodiments, the linker 100 is an oligomer, e.g., an oligomericlinker comprised of monomeric units. An oligomer, in some embodiments,comprises one or more types of monomeric units. Types of monomeric unitscan include, by way of example and not limitation, nucleotides (e.g.,ribonucleotides, deoxyribonucleotides, and analogs and derivativesthereof), amino acids (e.g., natural and unnatural amino acids),monosaccharides, and organic compounds such as phenyl- andalkynyl-containing compounds. In some embodiments, an oligomer comprisesone or more of the same type of monomeric unit. In some embodiments,oligomers comprised of one type of monomeric unit can be referred to asa polymer (e.g., a polymeric linker). In some embodiments, an oligomercomprises two or more different types of monomeric units (e.g., a mix ofmonomeric units).

In some embodiments, an oligomer (e.g., oligomeric linker or a polymericlinker) contains at least 5 monomeric units. In some embodiments, anoligomer contains at least 10 monomeric units. In some embodiments, anoligomer contains at least 10 and fewer than 200 monomeric units (e.g.,at least 10 and fewer than 150 monomeric units, at least 10 and fewerthan 100 monomeric units, at least 10 and fewer than 50 monomeric units,at least 10 and fewer than 40 monomeric units, at least 10 and fewerthan 30 monomeric units, or at least 10 and fewer than 20 monomericunits).

In some embodiments, the linker (e.g., polymeric linker, oligomericlinker) is a polysaccharide. Examples of polysaccharides suitable foruse as a linker 100 are known in the art (e.g., as described in SolidSupport Oligosaccharide Synthesis and Combinatorial CarbohydrateLibraries, Wiley 2001).

In some embodiments, the linker (e.g., polymeric linker, oligomericlinker) is a peptide. Non-limiting examples of peptides suitable for useas a linker 100 include, without limitation, oligopeptides, cyclicpeptides, and small proteins (e.g., avian pancreatic peptide-basedminiature proteins, such as described in Hodges, A. M. and Schepartz, A.(2007) J. Am. Chem. Soc. 129:11024-11025). Methods of engineeringgeometric constraints into peptide structure are well known in the artand are envisioned to be particularly useful, e.g., to impart rigidityand promote label separation. For example, proline content of a peptideamino acid sequence can be modified to control peptide shape (see, e.g.,Kritzer, J. A., et al. (2006) ChemBioChem 7:29-31). Additionalnon-limiting examples of useful peptide engineering techniques includepeptide cyclization (see, e.g., Maltsev, O. V., et al. (2016) AngewandteChemie 55(4):1535-1539), a-helical peptide constraint via staplingand/or H-bond surrogates (see, e.g., Douse, C. H., et al. (2014) ACSChem. Biol. 9:2204-2209), peptide constraint via cyclic (3-sheet and(3-hairpin mimics (see, e.g., Gibbs, A. C., et al. (1998) Nat. Struc.Biol. 5:284-288).

In some embodiments, the linker (e.g., polymeric linker, oligomericlinker) is a nucleic acid. In some aspects, a brightly labeled reactantcan be designed according to the diagram shown in FIG. 2A, whichgenerically depicts two luminescent labels attached to a core nucleicacid construct. As shown, in some embodiments, at least one luminescentlabel 210 is attached to a nucleic acid linker 200 at an attachment site202 via a spacer 220. In some embodiments, the nucleic acid linker 200comprises at least two hybridized oligonucleotide strands. In someembodiments, the nucleic acid linker 200 comprises at least twoluminescent labels, where each luminescent label is separated from thenext by a minimum distance d_(L). In some embodiments, at least two ofthe luminescent labels are attached to the same oligonucleotide strand,where each attachment site is separated from the next by a minimumdistance d_(A). In some embodiments, the luminescent labels are attachedto the oligonucleotide strand via spacers, where each spacer separates agiven luminescent label from its attachment site by a minimum distanced_(AL).

In some embodiments, the minimum distances d_(L), d_(A), and d_(AL) canbe obtained, for example, using theoretical methods known in the art(e.g., computationally or otherwise). In some embodiments, theoreticalmethods can include any approach that accounts for molecular structure,such as bond lengths, bond angles and rotation, electrostatics, nucleicacid helicity, and other physical factors which might be representativeof a molecule in solution. In some embodiments, distance measurementscan be obtained experimentally, e.g., by crystallographic orspectroscopic means.

Aspects of the disclosure relate, at least in part, to the discoverythat brightly labeled reactants (e.g., labeled nucleotides) having anucleic acid linker can be designed according to Equation 1:2(d _(AL))−d _(A)<12 Å,  Equation 1where 2(d_(AL))−d_(A) can be negative. In some embodiments, d_(A) isgreater than 17 angstroms (Å). In some embodiments, d_(A) is at least 17Å, but not greater than 350 Å (e.g., d_(A) is between about 17 and 350Å, between about 17 and 300 Å, between about 17 and 250 Å, between about17 and 200 Å, between about 17 and 150 Å, between about 17 and 100 Å, orbetween about 17 and 50 Å).

In yet other aspects, labeled nucleotides of the disclosure can bedesigned according to Equation 2:2(d _(AL))/d _(A)<1.  Equation 2

In some embodiments, 2(d_(AL))/d_(A) is less than 1, preferably lessthan 0.5. In some embodiments 2(d_(AL))/d_(A) is less than 0.1.

In some embodiments, labeled nucleotides of the disclosure can bedesigned according to Equation 3:[2(d _(AL))+LLD]/d _(A)<1,  Equation 3where LLD is a distance that represents the longest label dimension(LLD). In some embodiments, [2(d_(AL))+LLD]/d_(A) is less than 0.5. Insome embodiments, [2(d_(AL))+LLD]/d_(A) is less than 0.2. In someembodiments, [2(d_(AL))+LLD]/d_(A) is less than 0.1.

In some embodiments, minimum distance d_(AL) is measured from anapproximately central atom of a luminescent label 210 to an atom on theoligonucleotide strand to which the spacer 220 is attached. In someembodiments, minimum distance d_(AL) is measured from the center of theluminescent label 210 (e.g., approximated based on the center of mass ofthe luminescent molecule, or some other method known in the art ordescribed elsewhere herein) to an atom on the oligonucleotide strand towhich the spacer 220 is attached. In some embodiments, minimum distanced_(AL) is measured as the length of spacer 220 (e.g., measured from anatom of spacer 220 that attaches luminescent label 210 to an atom ofspacer 220 that attaches to nucleic acid 200). In some embodiments,minimum distance d_(A) is measured as the distance between atoms on theoligonucleotide backbone to which the spacers are covalently bound(e.g., between carbon atoms of abasic attachment sites). In someembodiments, minimum distance d_(A) is measured as the distance betweenthe labeled bases on the oligonucleotide strand (e.g., between atoms onnucleobases of basic attachment sites).

In some embodiments, minimum distance d_(L) is measured as the distancebetween approximately central atoms of adjacent luminescent labels. Insome embodiments, adjacent luminescent labels are separated by adistance d_(L) of approximately 6 angstroms. In some embodiments,distance d_(L) is at least 6 angstroms (e.g., at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, or at least 12angstroms). In some embodiments, minimum distance d_(L) is measured asan approximation, which may or may not factor in spacer configuration,spacer flexibility, or nucleic acid flexibility.

Among other aspects, the disclosure provides general strategies fordeveloping the distance d_(L) between luminescent labels such thatmultiply-labeled reactants can be engineered to have maximizedbrightness without compromising emission lifetime. In some embodiments,adjacent luminescent labels of sufficient proximity can interact suchthat a quenching effect occurs, resulting in diminished and/orinconsistent values for emission lifetime. Accordingly, the generaldesign strategies provided herein can, in some embodiments, involvestructural constraints that limit the extent of interactions betweenadjacent luminescent labels.

In some embodiments, a luminescent label can interact with guaninenucleobases of a nucleic acid linker via radiative and/or non-radiativedecay to effect diminished and/or inconsistent emission lifetime. Insome embodiments, luminescent label attachment sites are developed byminimizing G/C content in regions surround the attachment sites. Assuch, in some embodiments, attachment sites are located in A/T-richregions of an oligonucleotide strand. In some embodiments, eachattachment site is at least 2 nucleotides separated from a G or Cnucleotide on the oligonucleotide strand (e.g., 2, 3, 4, 5, 6, 7, 8, 9,or more than 10 nucleotides separated from a G or C nucleotide). Thus,in some embodiments, each attachment site is flanked by at least 2consecutive nucleotides selected from A or T.

In certain embodiments, the distance between attachment sites of anoligonucleotide strand can be described by the number of interveningunlabeled bases (e.g., intervening nucleotides). In some embodiments,attachment sites on an oligonucleotide strand are separated by at least5 unlabeled bases (e.g., at least 5, at least 6, at least 7, at least 8,at least 9, or at least 10 unlabeled bases). In some embodiments,attachment sites are separated by 6, 7, 8, or 9 unlabeled bases. In someembodiments attachment sites are separated by between 5 and 100unlabeled bases (e.g., between 5 and 80, between 5 and 60, between 5 and40, between 5 and 20, or between 5 and 10 unlabeled based) on theoligonucleotide strand.

In some embodiments, the design principles described herein allow forthe addition of successive luminescent labels to a labeled reactioncomponent for increasing brightness and/or luminescent intensity. Insome embodiments, techniques of the present application providemultiply-labeled reaction components having brightness and/orluminescent intensity according to the formula L_(n)(x), where L_(n) isequal to the total number of luminescent labels on a labeled reactantand x is equal to the measured brightness or fluorescent intensity ofthe corresponding singly-labeled reactant. Accordingly, in someembodiments, a two-dye labeled reaction component possesses brightnessand/or luminescent intensity that is doubled compared to the one-dyelabeled analog. In some embodiments, a three- or four-dye labeledreaction component possesses brightness and/or luminescent intensitythat is tripled or quadrupled, respectively, compared to the one-dyelabeled analog. In some embodiments, the brightly labeled reactantsdescribed herein exhibit brightness and/or luminescent intensity that isat least 70%, at least 80%, at least 90%, at least 95%, at least 98%, orat least 99% of the value predicted by L_(n)(x).

In some embodiments, the nucleic acid linkers provided herein comprisean oligonucleotide strand having at least two luminescent labels. FIG.2B generically depicts a single-stranded nucleic acid linker 250attached to two luminescent labels. As shown, a single-stranded linkercan possess a relatively high degree of flexibility that may promoteinteraction between labels. In some embodiments, brightly labeledreactants having consistent and/or preserved lifetime can be generatedusing single-stranded linker constructs, e.g., by utilizing certainlabels that do not interact to produce a quenching effect, such ascyanine-based dyes. However, for several classes of dyes, the promotionof label-label proximity due to linker flexibility may producediminished and/or inconsistent emission lifetimes. Accordingly, in someembodiments, the labeled oligonucleotide strand is hybridized with oneor more oligonucleotide strands to decrease overall nucleic acid linkerflexibility.

In some embodiments, strand hybridization was used as a general designstrategy to develop an appropriate distance d_(L) between luminescentlabels on a nucleic acid linker. In some embodiments, strandhybridization was used to increase rigidity in specific regions ofnucleic acids (e.g., within a labeled region and/or in a regionseparating a labeled region from a reactant). FIG. 2B genericallydepicts a double-stranded nucleic acid linker 252 that includes ahybridized oligonucleotide strand to rigidify the labeled region andthereby decrease strand flexibility that might otherwise promotelabel-label interaction.

As illustrated by nucleic acid linker 252, in some embodiments,luminescent label movement about an attachment point can promotelabel-label proximity. Also as shown, in some embodiments, labelmovement about an attachment site can result in a label being in closerproximity to an oligonucleotide strand of the nucleic acid linker. Insome embodiments, an oligonucleotide strand can interact with aluminescent label to produce inconsistent and/or diminished emissionlifetime measurements. Accordingly, in some embodiments, nucleic acidlinkers described herein comprise spacers having certain lengths,rigidities, sites of attachment, and/or configurations that limit theextent of label-label and/or linker-label interactions.

In some embodiments, separation distance between luminescent labels canbe defined in the context of the volume of space in which the labels arepresent relative to one another. For example, FIG. 2B depicts a diagram254 that illustrates an example of how distance d_(L) can be measured.In some embodiments, each luminescent label can be defined by a stericvolume 212. In some embodiments, steric volume 212 is approximated as asphere of radius 212-r or as an otherwise ellipsoidal shape. In someembodiments, each luminescent label comprises a steric volume 212 havinga center point that is at least 5 angstroms separated from that of anyother label (e.g., the center points of adjacent labels are separated byat least 5 angstroms). A center point of a luminescent label can be anysuitable center of the label, including, e.g., a center of mass or ageometric center of the label. In some embodiments, a center point of aluminescent label can be determined by calculating or approximatingradius 212-r, as illustrated in FIG. 2B. In some embodiments, a centerpoint of a luminescent label can be calculated or approximated asone-half of the longest dimension of a luminescent label (e.g., asdescribed elsewhere herein for r₁ and r₂ of FIG. 1A).

In some embodiments, distance between adjacent luminescent labels(d_(L)) can be measured as the distance between the centers of mass ofthe adjacent luminescent labels. Center of mass, in some embodiments,refers to the average position of all atoms in a luminescent label,weighted according to their masses. Methods of calculating center ofmass of are known in the art (see, e.g., Leach, A. R. MolecularModelling: Principles and Applications (2^(nd) edition), Prentice-Hall2001; Guenza, M. (2002) Macromolecules 35(7):2714-2722).

In some embodiments, distance between adjacent luminescent labels (dL)can be measured as the distance between the geometric centers of theadjacent luminescent labels. A geometric center of a molecule, in someembodiments, refers to the average position of all atoms of the molecule(e.g., all atoms in a luminescent label), wherein the atoms are notweighted. Thus, in some embodiments, the geometric center of a moleculerefers to a point in space that is an average of the coordinates of allatoms in the molecule.

In some embodiments, steric volume 212 is calculated more preciselyusing any suitable method known in the art. For example, molarrefractivity factors in properties that include polarizability, and canbe calculated according to the equation:Molar Refractivity=[(n ²−1)/(n ²+2)]×(MW/d),where n=index of refraction; MW=molecular weight; and d=density.

In some embodiments, steric volume 212 of a label can be calculatedcomputationally to include additional and/or more complex factors. Forexample, Verloop steric factor provides spatial dimensions of a moleculebased on bond angles, van der Waals radii, bond lengths, and possibleconformations (e.g., see Harper, K. C., et al. (2012) Nature Chemistry4, 366-374).

In some embodiments, luminescent label movement about an attachment sitemay be factored into the separation distance d_(L). As shown in FIG. 2B,the range of label movement about each attachment site can be defined,in some embodiments, based on spacer length and steric volume of thelabel. This theoretical range of movement is illustrated by dashedlines. It should be appreciated that, in some embodiments, a variety ofthe compositions and design strategies described herein canadvantageously limit the extent to which the range of movementapproaches the overlapping region shown. For example, in someembodiments, spacer rigidity, spacer length, and attachment siteseparation can each be addressed in accordance with the disclosure tolimit the extent to which the label is likely to approach theoverlapping region. It should also be appreciated that diagram 254 isintended for illustrative purposes, e.g., as labels are not necessarilyrequired to come into physical contact for radiative and/ornon-radiative decay to occur.

In some embodiments, the range of motion of labels depicted in diagram254 can be generally referred to as a spatial volume, e.g., an area ofspace having regions of varying probability that the label could bepresent at a point in space at a given point in time. In someembodiments, each label occupies a spatial volume that is substantiallynon-overlapping with that of any other luminescent label. In someembodiments, each label occupies a spatial volume that is substantiallyfree of any other label.

In some embodiments, relative attachment sites for luminescent labelscan be designed in view of the helical structure of a double-strandedlinker. FIG. 2C depicts an example of a nucleic acid 260 having twolabels separated by a distance, x. Nucleic acid 262 (drawn approximatelyto scale), is attached to two luminescent labels at attachment sitesseparated by roughly half as many bases as nucleic acid 260, but therelative attachment site locations along the helix result in a labelseparation distance through space of approximately 2x. As shown, in someembodiments, the nucleic acid linker can further limit the extent ofradiative and/or non-radiative decay between labels by acting as asteric barrier between labels.

In some embodiments, the term “steric barrier” refers to a linker or aportion of a linker (e.g., a nucleic acid linker or a portion thereof)positioned between a luminescent label attached to the linker and someother attachment of the linker. Without wishing to be bound by theory,it is thought that a steric barrier can absorb, deflect, or otherwiseblock radiative and/or non-radiative decay emitted by the luminescentlabel. In some embodiments, the steric barrier prevents or limits theextent to which one or more luminescent labels interact with one or moreother luminescent labels. In some embodiments, the steric barrierprevents or limits the extent to which one or more luminescent labelsinteract with one or more reactants. In some embodiments, the stericbarrier prevents or limits the extent to which one or more luminescentlabels interact with one or more molecules associated with a reactant(e.g., a polymerase bound to the reactant). Accordingly, in someembodiments, the term steric barrier can generally refer to a protectiveor shielding effect that is provided by some portion of a nucleic acidlinker.

In some embodiments, one or more structural motifs can function as asteric barrier. For example, in some embodiments, a helix formed by adouble-stranded nucleic acid linker functions as a steric barrier. Insome embodiments, a stem-loop or a portion thereof (e.g., a stem, aloop) functions as a steric barrier. In some embodiments, a three-wayjunction (e.g., as in a nucleic acid having two or more stem-loops)functions as a steric barrier. In some embodiments, a hybridized strandhaving no attachments (e.g., a support strand) functions as a stericbarrier. In some embodiments, a spacer functions as a steric barrier.

In some embodiments, the brightly labeled reactants described hereinprovide separation between luminescent labels and a reactant. In someembodiments, a nucleic acid 264 comprises a nucleoside polyphosphate 280reactant that serves as a substrate for a polymerase 290 in a synthesisreaction. In some embodiments, labels in close proximity to a polymeraseactive site can induce polymerase photodamage (e.g., via non-radiativedecay or otherwise), which can be detrimental to polymerase activity. Asshown, nucleic acid 266 is attached to a reactant such that at least aportion of the linker is in an intervening region between the label andthe reactant. As such, in some embodiments, a nucleic acid linker canfunction as a steric barrier to further protect the polymerase fromlabel-induced photodamage. This effect of polymerase protection can, insome embodiments, occur through label-reactant spatial separation and/orthe presence of a steric barrier between label and reactant. Polymeraseprotection is described in further detail in co-pending U.S. patentapplication Ser. No. 15/600,979), the content of which is incorporatedherein by reference in its entirety.

In some embodiments, the size and configuration of nucleic acid linkers(e.g., one or more oligonucleotide strands and/or one or more spacers ofa nucleic acid linker) determines the distance between a luminescentlabel and a nucleoside polyphosphate. In some embodiments, the distanceis about 1 nm or 2 nm to about 20 nm. For example, more than 2 nm, morethan 5 nm, 5-10 nm, more than 10 nm, 10-15 nm, more than 15 nm, 15-20nm, more than 20 nm. However, the distance between the luminescent labeland the nucleoside polyphosphate cannot be too long since certaindetection techniques require that the luminescent label be within adefined illumination volume to be excited (e.g., when the nucleosidepolyphosphate is held within the active site of a polymerase).Accordingly, in some embodiments, the overall distance is less than 30nm, less than 25 nm, around 20 nm, or less than 20 nm.

In some embodiments, other features of the compositions described hereincan be implemented to promote label-reactant separation to minimize thepotential for label-induced photodamage, e.g., spacer length, spacerrigidity, attachment site location. In some embodiments, reactantconnectivity to the nucleic acid linkers described herein can bemodified relative to a luminescent label to promote label-reactantseparation. FIG. 3A generically depicts nucleic acid linkers havingsame-strand or opposite strand label-reactant connectivity. In someembodiments, a nucleic acid 300 comprises two or more luminescent labelsand a reactant attached to the same oligonucleotide strand. In someembodiments, same-strand connectivity results in a covalent connectionbetween label and reactant.

In some embodiments, a nucleic acid 302 comprises two or moreluminescent labels and a reactant attached to different oligonucleotidestrands. In some embodiments, opposite strand connectivity results in anon-covalent connection between label and reactant. In some embodiments,opposite strand connectivity of label and reactant occurs throughhybridization of a label-attached oligonucleotide strand and areactant-attached oligonucleotide strand. As shown, in some embodiments,nucleic acid linkers of the disclosure comprise a label-reactantseparation distance d_(LR). In some embodiments, d_(LR) is the distancebetween reactant and the nearest luminescent label. In some embodiments,d_(LR) is measured from label attachment site to reactant attachmentsite. In some embodiments, d_(LR) is measured from the luminescentmolecule of a label to the reactant. In some embodiments, d_(LR) is atleast 1 nm. In some embodiments, d_(LR) is between about 1 nm to about10 nm (e.g., approximately 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8nm, 9 nm, 10 nm, or more than 10 nm). In some embodiments, d_(LR) isbetween about 2 nm to about 30 nm (e.g., between about 2 and 25 nm,between about 2 and 20 nm, between about 2 and 15 nm, between about 2and 10 nm, between about 2 and 5 nm, between about 5 and 10 nm, betweenabout 10 and 20 nm, between about 5 and 30 nm, between about 15 and 30nm, or between about 20 and 30 nm).

FIG. 3B depicts examples of non-limiting distance specifications used inthe design of brightly labeled reactants. In some embodiments, a nucleicacid linker 304 is attached to two luminescent labels and a nucleosidehexaphosphate (e.g., a reactant) via same-strand connectivity. In someembodiments, as shown, & is approximately 1 nm and d_(LR) isapproximately 7 nm. In some embodiments, the brightly labeled reactantsof the disclosure comprise more than two luminescent labels. Forexample, in some embodiments, a nucleic acid linker 306 is attached tothree luminescent labels and a nucleoside hexaphosphate (e.g., areactant) via same-strand connectivity. As shown, in some embodiments,constructs having more than two luminescent labels attached at more thantwo attachment sites will necessarily have more than one separationdistance d_(L). In some embodiments, each separation distance d_(L) on asingle construct can be independently designed according to thedescription herein. In some embodiments, each label separation distanced_(L) can be designed to be approximately the same. For example, in someembodiments, each occurrence of separation distance d_(L) isapproximately 3.5 nm and d_(LR) is approximately 7 nm.

In some embodiments, the brightly labeled reactants of the applicationcomprise two or more luminescent labels attached via differentoligonucleotide strands. For example, in some embodiments, a nucleicacid linker 308 comprises four luminescent labels attached via separateoligonucleotide strands. In some embodiments, an unlabeledoligonucleotide strand of nucleic acid linker 308 comprises a firstportion that is complementary to a first oligonucleotide strand attachedto two luminescent labels and a nucleoside polyphosphate. The unlabeledoligonucleotide strand further comprises a second portion that iscomplementary to a second oligonucleotide strand attached to twoluminescent labels. As shown, in some embodiments, luminescent labelattachment sites within each labeled strand are separated by 9nucleotides. In some embodiments, separation between nearest attachmentsites on non-contiguous labeled strands can be the same or different(e.g., as shown by a 10-nucleotide separation).

The non-limiting generic nucleic acid linker 304 having same-strandlabel-reactant connectivity was used as a basis for generating thelabeled nucleoside polyphosphate shown in FIG. 3C. As shown, the nucleicacid linker of FIG. 3C comprises two hybridized oligonucleotide strands.In accordance with certain embodiments of the disclosure, twoluminescent labels were attached at abasic sites within a firstoligonucleotide strand of the linker. Click chemistry techniques, asdescribed elsewhere herein as a general attachment strategy, were usedto attach a nucleoside polyphosphate to the same oligonucleotide strand.A second oligonucleotide strand was hybridized to the first strand topromote constrained spatial conformations of the attached components inaccordance with theory described herein. The construct of FIG. 3C wassuccessfully used in a single molecule sequencing run with other labelednucleoside polyphosphates (FIG. 3D). A similarly designed construct wasgenerated having opposite-strand connectivity and is shown in FIG. 3E.

FIG. 3E depicts an example structure of a nucleic acid linker thatnon-covalently connects a nucleoside polyphosphate (e.g., a reactant)with luminescent labels. As shown, a first oligonucleotide strandattached to two luminescent labels at abasic attachment sites washybridized with a second oligonucleotide strand attached to a nucleosidehexaphosphate at a terminal attachment site. In some embodiments, thedesign strategies provided in the present disclosure to generatebrightly labeled reactants is contemplated to include alternative labelconjugation strategies, such as labels integrated into a linker (e.g.,integrated into an oligomeric or polymeric linker, such as anoligonucleotide strand). For example, FIG. 3F depicts an example of anucleic acid linker having two labels attached within an oligonucleotidestrand.

As shown in FIG. 3F, in some embodiments, a brightly labeled reactantcomprises two luminescent labels attached within an oligonucleotidestrand (e.g., integrated into the strand). In some embodiments, suchconjugation strategies can be implemented with luminescent moleculesthat do not interact via radiative and/or non-radiative decay, such ascyanine-based dyes (e.g., as shown in FIG. 3F, boxed area). In someembodiments, the number of luminescent molecules attached within anoligonucleotide strand may be limited only by the size of theoligonucleotide strand. Thus, in some embodiments, a brightly labeledreactant comprises more than two luminescent labels (e.g., two, three,four, five, six, or more than six luminescent labels) attached within anoligonucleotide strand. The construct depicted in FIG. 3F wassuccessfully utilized in a sequencing run with three other brightlylabeled nucleoside polyphosphates (FIG. 3G).

The disclosure relates, in some aspects, to the discovery that nucleicacid linkers can be engineered having structurally-constrainedconformations that provide scaffolds for brightly labeled reactantshaving consistent and/or preserved emission lifetime. As describedherein, in some embodiments, oligonucleotide strand hybridization is onegeneral strategy that promotes constrained conformations for purposes oflabel separation. In some embodiments, oligonucleotide strandhybridization involves hybridization of different oligonucleotidestrands. In some embodiments, oligonucleotide strand hybridizationinvolves self-strand hybridization (e.g., self-hybridizing within asingle strand). In some embodiments, self-strand hybridization may limitnucleic acid linker flexibility, as described elsewhere herein withregard to separate strand hybridization. In some embodiments,self-strand hybridization is used to form one or more stem-loopstructures in a nucleic acid linker.

A stem-loop, or hairpin loop, is an unpaired loop of nucleotides on anoligonucleotide strand that is formed when the oligonucleotide strandfolds and forms base pairs with another section of the same strand. Insome embodiments, the unpaired loop of a stem-loop comprises three toten nucleotides. Accordingly, a stem-loop can be formed by two regionsof an oligonucleotide strand having inverted complementary sequencesthat hybridize to form a stem, where the two regions are separated bythe three to ten nucleotides that form the unpaired loop. In someembodiments, the stem can be designed to have one or more G/Cnucleotides, which can provide added stability with the additionhydrogen bonding interaction that forms compared to A/T nucleotides. Insome embodiments, the stem comprises G/C nucleotides immediatelyadjacent to an unpaired loop sequence. In some embodiments, the stemcomprises G/C nucleotides within the first 2, 3, 4, or 5 nucleotidesadjacent to an unpaired loop sequence.

In some embodiments, an unpaired loop of a stem-loop nucleic acid linkercomprises one or more luminescent labels. Thus, in some embodiments, oneor more label attachment sites are present in an unpaired loop. In someembodiments, an attachment site occurs at an abasic site in the unpairedloop. In some embodiments, an attachment site occurs at a base of theunpaired loop. In some embodiments, a loop comprises an A/T/U-richsequence, e.g., due to a quenching effect observed with guanine, asdescribed elsewhere herein. In some embodiments, the attachment siteoccurs at an A, T, or U nucleotide in the unpaired loop. In someembodiments, at least four consecutive A, T, or U nucleotides occur oneither side of an attachment site. In some embodiments, an unpaired loopcomprises less than 33% G/C content (e.g., less than 30%, less than 20%,less than 10%, or 0% G/C content).

In some embodiments, a luminescent label is attached to an unpaired loopof a stem-loop (e.g., the label attachment site occurs within the loop).For example, FIG. 4A generically depicts an example of a labeledreactant having a stem-loop nucleic acid linker 400. As shown, in someembodiments, a first oligonucleotide strand 410 self-hybridizes to forma stem-loop. In some embodiments, the self-hybridized portion of theoligonucleotide strand forms a stem 412 of the stem-loop. In someembodiments, self-strand hybridization results in the formation of aloop 414 of a stem-loop (e.g., an unpaired loop). In some embodiments,the first oligonucleotide strand 410 is further hybridized with a secondoligonucleotide strand 420 attached to a reactant. Thus, in someembodiments, a stem-loop nucleic acid linker non-covalently connects aluminescent label to a reactant via opposite strand connectivity. Itshould be appreciated that, in some embodiments, a self-hybridizedoligonucleotide strand (e.g., a strand having a stem-loop) attached to aluminescent label can be attached to one or more reactants viasame-strand connectivity.

In some embodiments, two or more luminescent labels are attached to astem-loop nucleic acid linker. For example, in some embodiments, astem-loop nucleic acid linker 402 is attached to two luminescent labels.As shown, in some embodiments, attachment sites for the two luminescentlabels occur within the loop of the stem-loop. In some embodiments, thestability provided by the self-hybridized stem region results in theloop region having a relatively low level of flexibility. In someembodiments, luminescent label attachment sites within an unpaired loopcan be designed to promote a favorable label-label spatial separationand/or a favorable label-reactant spatial separation. The exampleprovided by the non-limiting stem-loop nucleic acid linker 402 depictslabel attachment sites that are separated from one another by a distanceapproximating the diameter of the loop. In some embodiments, this designcan maximize label-label separation through space. In some embodiments,this design further limits the extent of label-label interaction througha steric barrier effect provided by the loop, as described elsewhereherein. In some embodiments, a stem-loop nucleic acid linker comprisestwo or more stem-loops (FIG. 4B).

As shown in FIG. 4B, in some embodiments, a stem-loop nucleic acidlinker 404 comprises two stem-loops. In some embodiments, formation oftwo stem-loops results in a nucleic acid linker having a Y-shape suchthat a three-way junction 410 is formed. In some embodiments, three-wayjunctions are contemplated as a general design strategy due to therelative stability and geometrically constrained conformation thatresults from these features. In some embodiments, a stem-loop nucleicacid linker 404 comprises one luminescent label attached to each loop.In some embodiments, a stem-loop nucleic acid 404 non-covalentlyconnects a luminescent label and a reactant through opposite strandconnectivity, e.g., one or more labels attached to a firstoligonucleotide strand hybridized with a second oligonucleotide strandattached to one or more reactants. In some embodiments, a stem-loopnucleic acid 406 connects a luminescent label and a reactant viasame-strand connectivity (e.g., one or more labels and one or morereactants attached to the same oligonucleotide strand). Stem-loopnucleic acid 406 depicts an example of non-limiting distancespecifications used in the design of brightly labeled reactants having athree-way junction. For example, these approximate distances were usedas a basis for generating the labeled nucleoside polyphosphate shown inFIG. 4C. The construct of FIG. 4C was successfully used in a singlemolecule sequencing run with other labeled nucleoside polyphosphates(FIG. 4D).

In some embodiments, one or more luminescent labels are attached to astem of a stem-loop. In some embodiments, a stem-loop of a nucleic acidlinker does not comprise a luminescent label. In some embodiments, astem-loop of a nucleic acid linker comprises an unpaired region withinthe stem (e.g., a “bulge loop”). In some embodiments, one or moreunlabeled structural motifs, such as stem-loops, are included in thenucleic acid linker (e.g., in a position on the nucleic acid linker suchthat a that is between the one or more luminescent labels and the one ormore nucleoside polyphosphates). In such embodiments, the one or moreunlabeled structural motifs can provide a steric barrier effect, asdescribed elsewhere herein.

In some embodiments, a stem-loop nucleic acid linker comprises a firstoligonucleotide strand attached to a luminescent label. In someembodiments, the luminescent label is attached at an attachment site onthe first oligonucleotide strand via a spacer. In some embodiments, thefirst oligonucleotide strand forms a stem-loop secondary structurehaving a stem and a loop (e.g., an unpaired loop). In some embodiments,the loop comprises the attachment site. In some embodiments, the nucleicacid linker comprises a second oligonucleotide strand hybridized withthe first oligonucleotide strand. In some embodiments, the secondoligonucleotide strand is attached to a nucleoside polyphosphate. Insome embodiments, the nucleoside polyphosphate is attached to the secondoligonucleotide strand via a spacer. In some embodiments, the firstoligonucleotide strand is of the structure:

wherein N_(A) and N_(B) are each a consecutive sequence of 5 to 40nucleotides independently selected from A, U, T, G, and C, wherein thesecond oligonucleotide strand is hybridized to either a 5′ portion ofN_(A) or a 3′ portion of N_(B); brackets denote a region that forms ystem-loops, each stem-loop having a stem and a loop, wherein y is 1 to3; N₁ and N₂ are each a consecutive sequence of 5 to 20 nucleotidesindependently selected from A, U, T, G, and C, wherein: a 5′ portion ofN₁ (S₁) and a 3′ portion of N₂ (S₂) are reverse complementary orpartially reverse complementary and are capable of hybridizing with oneanother to form the stem motif; a 3′ portion of N₁ (L₁), a 5′ portion ofN₂ (L₂), and an intervening region form the loop motif when S₁ and S₂hybridize to form the stem motif; (A/T) is a nucleotide selected from A,T, and U; X₁ is the attachment site on the second oligonucleotidestrand; Y₁ is the first linker; and Z₁ is the luminescent label.

As described herein, aspects of the disclosure relate to geometricallyconstrained linker configurations for connecting one or more luminescentlabels to one or more reactants (e.g., one or more nucleosidepolyphosphates). In some embodiments, a geometrically constrainedconfiguration refers to a nucleic acid linker having two or moreluminescent labels, where the luminescent labels are spatially separatedby a symmetrical configuration. In some aspects, the brightly labeledreactants of the disclosure comprise nucleic acid linkers resembling atree-shaped configuration as depicted in FIG. 5A. In some embodiments,tree-shaped linker 500 comprises a tris-labeled nucleic acid linkerconnected to a reactant (e.g., a nucleoside polyphosphate). As shown, insome embodiments, tree-shaped linker 500 comprises three mainoligonucleotide components. In some embodiments, a first oligonucleotidecomponent 510 comprises four oligonucleotide strands covalently attachedvia a branched linker. In some embodiments, three of the oligonucleotidestrands of component 510 are each attached to a luminescent label (e.g.,via terminal attachment or other conjugation strategy described herein).Accordingly, in some embodiments, the three labeled oligonucleotidestrands of component 510 are generally referred to as a labeled portionof component 510. In some embodiments, a fourth oligonucleotide strandof component 510 is unlabeled. In some embodiments, the fourtholigonucleotide strand is hybridized with a second oligonucleotidecomponent 520. In some embodiments, the second oligonucleotide component520 of nucleic acid linker 500 is attached to a reactant (e.g., viaterminal attachment or other conjugation strategy described herein). Insome embodiments, a third oligonucleotide component 530 is hybridizedwith the three oligonucleotide strands of the labeled portion ofcomponent 510. In some embodiments, the third oligonucleotide component530 is referred to as a support strand, as its hybridization withcomponent 510 provides structural rigidity in the labeled region topromote spatial separation of the luminescent labels.

As should be appreciated, any of the linker design strategies describedherein can be applied to a tree-shaped nucleic acid linker or any othernucleic acid linker configuration contemplated by the disclosure (e.g.,strand connectivity, spacer properties and spacer configurations,label-label separation, label-reactant separation, three-way junctions,etc.). Tree-shaped nucleic acid 502 depicts an example of non-limitingdistance specifications used in the design of brightly labeled reactantshaving a tree-shaped nucleic acid linker. As an illustrative example,these approximate distances were used as a basis for generating thelabeled nucleoside polyphosphate shown in FIG. 5B. As shown, in someembodiments, a tree-shaped nucleic acid can comprise more than onereactant (e.g., two nucleoside polyphosphates, or more than twonucleoside polyphosphates).

In some embodiments, a tree-shaped nucleic acid linker is described interms of first and second oligonucleotide strands that comprise thehybridized portion having a reactant. For example, in some embodiments,a tree-shaped nucleic acid linker comprises a first oligonucleotidestrand attached to two or more branching oligonucleotide strands at aterminal end of the first oligonucleotide via a coupling compound (e.g.,a branched coupler). In some embodiments, each branching oligonucleotidestrand is attached to a luminescent label. In some embodiments, thefirst oligonucleotide strand is hybridized with a second oligonucleotidestrand attached to a reactant (e.g. a nucleoside polyphosphate). In someembodiments, a third oligonucleotide strand (e.g., a thirdoligonucleotide component) is hybridized with the two or more branchingoligonucleotide strands. In some embodiments, the coupling compound isof the below structure:

wherein N_(f) is the first oligonucleotide strand; N_(b) is a branchingoligonucleotide strand; R_(f) and R_(b) are each, independent from oneanother, a bond or a linking group selected from the group consisting ofsubstituted or unsubstituted alkylene; substituted or unsubstitutedalkenylene; substituted or unsubstituted alkynylene; substituted orunsubstituted heteroalkylene; substituted or unsubstitutedheteroalkenylene; substituted or unsubstituted heteroalkynylene;substituted or unsubstituted heterocyclylene; substituted orunsubstituted carbocyclylene; substituted or unsubstituted arylene;substituted or unsubstituted heteroarylene; and combinations thereof;and each instance of O is an oxygen atom of either a 5′ phosphate groupor a 3′ hydroxyl group of an adjacent oligonucleotide strand.

In some aspects, the disclosure provides brightly labeled reactantshaving star-shaped nucleic acid linkers. As shown in FIG. 6A,star-shaped nucleic acid linkers can provide symmetrically-arrangedreactants at an external region of the linker and one or moreluminescent labels nearer to a core region of the configuration. In thisway, in some embodiments, reactants may be more susceptible to reacting,e.g., with a polymerase. In some embodiments, the nucleotide contentsurrounding the luminescent label attachment sites near the core regionare selected according to specifications described herein (e.g., low G/Ccontent to avoid a quenching effect between a label and the linker). Insome embodiments, a star-shaped nucleic acid linker 600 comprises aY-shaped oligonucleotide component having three oligonucleotide strandsattached via a branched linker. In some embodiments, each of the threeoligonucleotide strands of the Y-shaped oligonucleotide component isattached (e.g., terminally) to a reactant. In some embodiments, each ofthe three oligonucleotide strands of the Y-shaped oligonucleotidecomponent is hybridized with an oligonucleotide strand. In someembodiments, one of the hybridized oligonucleotide strands is attachedto a luminescent label. In some embodiments, however, two or three ofthe hybridized oligonucleotide strands are each attached to aluminescent label.

In some embodiments, the Y-shaped oligonucleotide component of astar-shaped nucleic acid linker is attached to more than threereactants. For example, in some embodiments, a brightly labeled reactanthaving a star-shaped nucleic acid linker 602 comprises two reactants ateach of two oligonucleotide strands of the Y-shaped oligonucleotidecomponent. In some embodiments, the two reactants are attached tostrands of the Y-shaped oligonucleotide component that are hybridizedwith unlabeled strands, as shown.

In some embodiments, a star-shaped nucleic acid linker comprises alabeled oligonucleotide component and a reactant oligonucleotidecomponent, where each oligonucleotide component comprises a Y-shapedoligonucleotide. For example, in some embodiments, a star-shaped nucleicacid linker 604 comprises a first Y-shaped oligonucleotide componentattached to three luminescent labels. In some embodiments, eachluminescent label is attached to each oligonucleotide strand of thefirst Y-shaped component. In some embodiments, as shown, eachluminescent label is attached near a core region of the first Y-shapedoligonucleotide component. In some embodiments, star-shaped nucleic acidlinker 604 is hybridized with a second Y-shaped oligonucleotidecomponent attached to three reactants. In some embodiments, eachreactant is attached to each oligonucleotide strand of the secondY-shaped component. In some embodiments, as shown, each reactant isattached at an external region of the first Y-shaped oligonucleotidecomponent (e.g., via terminal attachment sites). Star-shaped nucleicacid 606 depicts an example of non-limiting distance specifications usedin the design of brightly labeled reactants having a star-shaped nucleicacid linker. An example structure of a brightly labeled reactant havinga star-shaped nucleic acid linker generated in accordance with thedisclosure is shown in FIG. 6B.

In some embodiments, a star-shaped nucleic acid linker can be describedin terms of a covalent coupling compound used in a Y-shapedoligonucleotide component of the linker. For example, in someembodiments, a brightly labeled reactant having a star-shaped nucleicacid linker comprises a first oligonucleotide component that comprisesthree or more oligonucleotide strands extending from a covalent couplingcompound. In some embodiments, at least one of the oligonucleotidestrands is attached to a reactant (e.g., a nucleoside polyphosphate). Insome embodiments, two or more (e.g., 2, 3, 4, 5 or more) of theoligonucleotide strands of the first oligonucleotide component are eachattached to one or more reactants. In some embodiments, the firstoligonucleotide component is hybridized with a second oligonucleotidecomponent. In some embodiments the second oligonucleotide componentcomprises at least one oligonucleotide strand attached to a luminescentlabel. In some embodiments, the second oligonucleotide componentcomprises three or more oligonucleotide strands extending from acovalent coupling compound. In some embodiments, two or more (e.g., 2,3, 4, 5 or more) of the oligonucleotide strands of the secondoligonucleotide component are each attached to a luminescent label.

In some aspects, the brightly labeled reactants of the disclosurecomprise a nucleic acid linker having a tetrahedral core. As illustratedby the example structure of FIG. 6C, a tetrahedral core refers to afour-way covalent coupling compound that promotes asymmetrically-constrained spatial arrangement of one or more luminescentlabels and one or more nucleoside polyphosphates. FIG. 6D depicts anexample of a reaction scheme that can be used to synthesize the examplestructure shown in FIG. 6C. In some embodiments, a tetrahedral core iscontemplated for use in combination with any of one or more of thebrightly labeled reactant design strategies described herein. As anexample, the symmetrically-constrained benefits provided by atetrahedral core was implemented with three-way junctions describedherein to generate the brightly labeled reactant shown in FIG. 6E. Asshown, in some embodiments, attachment of a luminescent label at or neara three-way junction can advantageously provide a steric effect, asdescribed elsewhere herein.

In some embodiments, the disclosure contemplates furthersymmetrically-constrained configurations using core chemical couplersthat allow for symmetrical attachment. In some embodiments, for example,a brightly labeled reactant comprises a cyclodextrin-based core. Anexample synthetic scheme for generating a cyclodextrin-based nucleicacid linker having three-way junctions is shown in FIG. 7 . An examplestructure of a cyclodextrin coupling compound is provided in Table 1along with examples of other coupling compounds that may be used forcovalent linkage of three or more oligonucleotide strands in accordancewith embodiments described herein.

TABLE 1 Examples of coupling compounds for linking oligonucleotidestrands

Spacers

As described herein, in some embodiments, luminescent labels and/orreactants can be attached to a nucleic acid linker via a spacer. In someembodiments, the spacer attaches to an oligonucleotide strand of thenucleic acid linker at an attachment site. In some embodiments, theattachment site occurs at a terminal site on the oligonucleotide strand(e.g., at a 5′ or 3′ end). Examples of terminal attachment sites areprovided in the instant application, e.g., as shown in FIGS. 3C, 3E, 4C,6B, 6C, 8, 9, and 10 (terminal attachment of nucleotides), and in FIGS.5B, 9, and 10 (terminal attachment of luminescent labels). In someembodiments, the attachment site occurs at an abasic site within theoligonucleotide strand (e.g., at a site that lacks but is adjacent tonucleotides). Examples of abasic attachment sites are provided in theinstant application, e.g., as shown in FIGS. 3C, 3E, 6B, 6C, and 8(abasic attachment of luminescent labels). In some embodiments, theattachment site occurs at a basic site on the oligonucleotide strand(e.g., attached to a nucleotide, such as the nucleobase, sugar, orphosphate of a nucleotide on the strand). Examples of basic attachmentsites are provided in the instant application, e.g., as shown in FIGS.4C and 8 (basic attachment of luminescent labels).

In some embodiments, a spacer comprises a plurality of thymidinenucleotides. In some embodiments, the spacer comprises a branchedspacer, e.g., a branched thymidine spacer. In some embodiments, thebranched spacer comprises a branched thymidine spacer. For example, insome embodiments, each nucleoside polyphosphate comprises a thymidinespacer of the formula Nu-T(T)_(n)T-R, where Nu represents a nucleosidepolyphosphate, T represents a thymidine nucleotide, n is an integer witha value between 1 and 30, and R represents a point of convergenceconnecting one or more additional nucleoside polyphosphates. In someembodiments, the point of convergence is further attached directly to anoligonucleotide strand of the nucleic acid. In some embodiments, thepoint of convergence is further attached indirectly to theoligonucleotide strand, e.g., through further thymidine linkers and/orfurther points of convergence. An example of a branched thymidine spaceris shown in FIG. 5B, which depicts two nucleoside polyphosphates havingthymidine spacers and a point of convergence that is further attacheddirectly to an oligonucleotide strand.

In some embodiments, a spacer contains one or more points of divergenceso that two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)nucleoside polyphosphates are connected to each luminescent label, twoor more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) luminescent labelsare connected to each nucleoside polyphosphate, or two or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, or more) nucleoside polyphosphates areconnected to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)luminescent labels.

In some embodiments, the spacer is attached at a terminal site on anoligonucleotide strand of a nucleic acid linker. In some embodiments, aluminescent label and/or a reactant is attached at either a 5′ or 3′ endof an oligonucleotide strand via the generic spacer shown below:

where

is a luminescent label (or, in an alternative embodiment, a reactant);m>1; n>1; and m+n<10.

In some embodiments, the spacer is attached at an internal abasic sitewithin an oligonucleotide strand of a nucleic acid linker. In someembodiments, a luminescent label and/or a reactant is attached at aninternal abasic site on an oligonucleotide strand via the generic spacershown below:

where

is a luminescent label (or, in an alternative embodiment, a reactant);n>1; and X=CH₂ or O.

Examples of spacers for luminescent label attachment (e.g., viaterminal, internal abasic, or basic attachment sites) are provided inTable 2 and shown below. Although spacer structures may be shown havingluminescent labels, it should be appreciated that, in some embodiments,a reactant can be substituted such that any of the spacer structuresprovided herein can be used to attach a reactant (e.g., a nucleosidepolyphosphate) to a nucleic acid linker.

In some embodiments, a luminescent label and/or a reactant is attachedat an internal abasic site on an oligonucleotide strand via theglycolamine spacer shown below:

where

is a luminescent label (or, in an alternative embodiment, a reactant).

In some embodiments, a luminescent label and/or a reactant is attachedat an internal abasic site on an oligonucleotide strand via theserinolamine spacer shown below:

where

is a luminescent label (or, in an alternative embodiment, a reactant).

In some embodiments, a luminescent label and/or a reactant is attachedat an internal basic site on an oligonucleotide strand via theC6-amino-T spacer shown below:

where

is a luminescent label (or, in an alternative embodiment, a reactant).

In some embodiments, a luminescent label and/or a reactant is attachedat an internal basic site on an oligonucleotide strand via theC2-amino-T spacer shown below:

where

is a luminescent label (or, in an alternative embodiment, a reactant).

In some embodiments, a luminescent label and/or a reactant is attachedat an internal basic site on an oligonucleotide strand via theC8-alkyne-dT spacer shown below:

where

is a luminescent label (or, in an alternative embodiment, a reactant).

In some embodiments, one or more luminescent labels and/or one or morereactants (e.g., one or more nucleoside polyphosphates) can be attachedto a nucleic acid linker using chemical coupling techniques known in theart. For example, in some embodiments, click chemistry techniques (e.g.,copper-catalyzed, strain-promoted, copper-free click chemistry, etc.)can be used to attach the one or more luminescent labels and the one ormore nucleoside polyphosphates to the nucleic acid. In some embodiments,a nucleoside polyphosphate is coupled to a nucleic acid linker via anazide-conjugated dN6P (e.g., dN6P-N₃), according to the genericstructure shown below:

where Base is a nucleobase selected from adenine, cytosine, guanine,thymine, uracil, and derivatives thereof; —Y—Z—=−CH₂CH₂—, —CONH—, or—NHCO—; and X=NH or O. For example, in some embodiments, anazide-conjugated dN6P (e.g., dN6P-N₃) and/or an azide-conjugatedluminescent label (e.g., dye-N₃) is attached to a nucleic acid linker ina copper-catalyzed reaction by contacting the azide of dN6P-N₃ or dye-N₃with a terminal alkyne of an alkyne-conjugated nucleic acid linker undersuitable reaction conditions to form a triazole linkage between thenucleic acid linker and dN6P or dye. In some embodiments, anazide-conjugated dN6P (e.g., dN6P-N₃) and/or an azide-conjugatedluminescent label (e.g., dye-N₃) is attached to a nucleic acid linker ina copper-free reaction by contacting the azide of dN6P-N₃ or dye-N₃ withan internal alkyne of a cyclooctyne-conjugated nucleic acid linker undersuitable reaction conditions to form a multicyclic linkage between thenucleic acid linker and dN6P or dye. Cyclooctyne modification of anucleic acid linker can be accomplished using cyclooctyne reagentssuitable for generating copper-free click chemistry moieties known inthe art. For example, a cyclooctyne-modified nucleic acid linker isprepared by contacting a suitable cyclooctyne reagent (e.g.,(1R,8S,9s)-Bicyclo[6.1.0]non yn-9-ylmethyl N-succinimidyl carbonate)with a terminal amine of a nucleic acid linker.

Accordingly, in some embodiments, a spacer includes a coupled group(e.g., BCN, tetrazine, tetrazole, and other products generated via thecoupling of reactive moieties suitable for click reactions and similarcoupling techniques). In some embodiments, the spacer is of the formula:

wherein R₁ is a first linking group and is attached to the attachmentsite on the first oligonucleotide strand; R₂ is a second linking groupand comprises a coupled moiety formed in a coupling reaction performedto covalently join R₁ and R₃; and R₃ is a third linking group and isattached to the luminescent label.

In some embodiments, the spacer is of a formula selected from:

wherein R₁ and R₃ are each independently a bond or a linking groupselected from the group consisting of substituted or unsubstitutedalkylene; substituted or unsubstituted alkenylene; substituted orunsubstituted alkynylene; substituted or unsubstituted heteroalkylene;substituted or unsubstituted heteroalkenylene; substituted orunsubstituted heteroalkynylene; substituted or unsubstitutedheterocyclylene; substituted or unsubstituted carbocyclylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; and combinations thereof.

In some embodiments, examples of spacers generated using click chemistryare provided in Table 2, where

is a luminescent label (or, in an alternative embodiment, a reactant).

TABLE 2 Examples of clicked spacers

As used herein, a “nucleic acid linker” generally refers to a nucleicacid connecting one or more luminescent labels to one or more nucleosidepolyphosphates. In some embodiments, a nucleic acid linker can generallyrefer to a construct having any number of oligonucleotide strandsconnected through covalent attachment or through base-pairing (e.g.,hybridization). In some embodiments, a linker may be alternativelyreferred to as a “core” or a “base” construct to which differentfunctional components (e.g., one or more luminescent labels, one or morenucleoside polyphosphates) can be attached. The term “construct” is, insome embodiments, used throughout in various contexts to generally referto a linker, and may or may not encompass the various other componentsdescribed herein (e.g., spacer, label, nucleoside polyphosphate, etc.).

In some embodiments, the nucleic acid linkers described herein are notattached to a particle of material (e.g., are not attached to a particleof metallic, magnetic, polymeric, or other material). In someembodiments, a nucleic acid linker is a linear molecule (e.g., a“rod-shaped” nucleic acid). In some embodiments, a nucleic acid linkeris a circular molecule. In some embodiments, a nucleic acid linker issingle-stranded (e.g., with or without stem-loop structures). In someembodiments, a nucleic acid linker is double-stranded (e.g., with orwithout stem loop structures). In some embodiments, the two strands of adouble stranded nucleic acid linker are hybridized (due to complementarysequences) and not covalently attached. However, in some embodiments,one or more covalent bonds may be introduced (e.g., using one or morechemical linkers) to covalently attach two strands of a double strandedlinker. In some embodiments, a nucleic acid linker may include one ormore additional moieties as described herein. In some embodiments, anucleic acid linker includes i) one or more additional moieties withinor at the end(s) of the sugar phosphate backbone, ii) one or moremodifications (e.g., one or more modified bases or sugars), or acombination of i) and ii). However, in some embodiments a nucleic acidlinker does not include i), ii), or either of i) or ii).

It should be understood that, in the context of a nucleic acid linker, a“nucleotide” or “nucleoside polyphosphate” attached thereto refers tothe one or more nucleotides (e.g., nucleoside polyphosphates) that areconfigured to be incorporated into a growing nucleic acid strand (e.g.,during a sequencing reaction). In some embodiments, the one or morenucleotides comprise one or more nucleoside monophosphates or nucleosidepolyphosphates. Examples of nucleoside polyphosphates include, in someembodiments, nucleoside di- or triphosphates, or nucleosides with morethan three 5′ phosphates, such as nucleoside hexaphosphates.Accordingly, in some embodiments, a “labeled nucleotide” refers to anucleoside polyphosphate connected to one or more luminescent labelsthrough a linker of the application, where the nucleoside polyphosphateacts as a substrate for a polymerase enzyme under nucleic acid synthesisreaction conditions. In some embodiments, the nucleoside polyphosphatecomprises at least a diphosphate group or a triphosphate group which canbe acted upon by a suitable polymerase enzyme in a phosphoryl transferreaction (e.g., transfer of the a-phosphate of the nucleosidepolyphosphate from its (3-phosphate to the 3′ hydroxyl group of agrowing nucleic acid strand).

In some embodiments, the one or more nucleoside phosphates (e.g.,nucleoside polyphosphates) may be attached through a terminal phosphateto an oligonucleotide (e.g., a labeled or an unlabeled oligonucleotidestrand) that forms part of a linker of the application, which canfunction as a protecting molecule that protects a polymerase fromlabel-induced photodamage (e.g., as described elsewhere in thisapplication). In some embodiments of any of the compositions or methodsdescribed in this application, a phosphate portion (e.g., apolyphosphate portion) of a nucleoside phosphate (e.g., of a nucleosidepolyphosphate) includes one or more phosphates (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphate groups) or variants thereof. For example,in some embodiments, a phosphate portion (e.g., a polyphosphate portion)of a nucleoside phosphate (e.g., of a nucleoside polyphosphate) caninclude a phosphate ester, a thioester, a phosphoramidate, an alkylphosphonate linkage, other suitable linkage, or more than one suchmodifications, or a combination of two or more thereof. In someembodiments, the labeled and unlabeled strands of a nucleic acid linkerare substantially complementary to one another (e.g., over the length ofa dimerization domain wherein the strands within the dimerization domaincan have, for example, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% complementary to one another).

A nucleoside polyphosphate can have n phosphate groups, where n is anumber that is greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of nucleoside polyphosphates include nucleoside diphosphate andnucleoside triphosphate. A labeled nucleotide can be a terminalphosphate labeled nucleoside polyphosphate, such that a terminalphosphate of the nucleoside polyphosphate is attached to a linker (e.g.,a nucleic acid linker) that comprises one or more luminescent labels,thereby forming a labeled nucleoside polyphosphate. Such label can be aluminescent (e.g., fluorescent or chemiluminescent) label, a fluorogeniclabel, a colored label, a chromogenic label, a mass tag, anelectrostatic label, or an electrochemical label. A label (or marker)can be coupled to a terminal phosphate through a linker, such as aspacer as described herein. The linker (e.g., spacer) can include, forexample, at least one or a plurality of hydroxyl groups, sulfhydrylgroups, amino groups or haloalkyl groups, which may be suitable forforming, for example, a phosphate ester, a thioester, a phosphoramidateor an alkyl phosphonate linkage at the terminal phosphate of a naturalor modified nucleotide. A linker (e.g., a spacer) can be cleavable so asto separate a label from the terminal phosphate, such as with the aid ofa polymerization enzyme. Examples of nucleotides and linkers (e.g.,spacers) are provided in U.S. Pat. No. 7,041,812, which is entirelyincorporated herein by reference.

A nucleotide (e.g., a nucleoside polyphosphate) can comprise any of anadenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), orvariants thereof. A nucleotide (e.g., a nucleoside polyphosphate) cancomprise a methylated nucleobase. For example, a methylated nucleotidecan be a nucleotide that comprises one or more methyl groups attached tothe nucleobase (e.g., attached directly to a ring of the nucleobase,attached to a substituent of a ring of the nucleobase). Exemplarymethylated nucleobases include 1-methylthymine, 1-methyluracil,3-methyluracil, 3-methylcytosine, 5-methylcytosine, 1-methyladenine,2-methyladenine, 7-methyladenine, N₆-methyladenine,N6,N6-dimethyladenine, 1-methylguanine, 7-methylguanine,N2-methylguanine, and N2,N2-dimethylguanine.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenine (A), cytosine (C), guanine(G), thymine (T), and uracil (U), or variants thereof. In some examples,a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),or derivatives thereof. In some embodiments, the nucleic acid is amodified nucleic acid, including, without limitation, a locked nucleicacid (LNA), a peptide nucleic acid (PNA), a triazole-linked nucleicacid, a 2′-F-modified nucleic acid, and derivatives and analogs thereof.A nucleic acid may be single-stranded or double stranded. In someembodiments, a nucleic acid generally refers to any polymer ofnucleotides.

In some embodiments, the disclosure provides new compositions foridentifying single molecules based on one or more luminescent propertiesof those molecules. In some embodiments, a molecule (e.g., aluminescently labeled nucleoside polyphosphate) is identified based onits brightness, luminescent lifetime, absorption spectra, emissionspectra, luminescent quantum yield, luminescent intensity, or acombination of two or more thereof. Identifying may mean assigning theexact molecular identity of a molecule, or may mean distinguishing ordifferentiating the particular molecule from a set of possiblemolecules. In some embodiments, a plurality of single molecules can bedistinguished from each other based on different brightnesses,luminescent lifetimes, absorption spectra, emission spectra, luminescentquantum yields, luminescent intensities, or combinations of two or morethereof. In some embodiments, a single molecule is identified (e.g.,distinguished from other molecules) by exposing the molecule to a seriesof separate light pulses and evaluating the timing or other propertiesof each photon that is emitted from the molecule. In some embodiments,information for a plurality of photons emitted sequentially from asingle molecule is aggregated and evaluated to identify the molecule. Insome embodiments, a luminescent lifetime of a molecule is determinedfrom a plurality of photons that are emitted sequentially from themolecule, and the luminescent lifetime can be used to identify themolecule. In some embodiments, a luminescent intensity of a molecule isdetermined from a plurality of photons that are emitted sequentiallyfrom the molecule, and the luminescent intensity can be used to identifythe molecule. In some embodiments, a luminescent lifetime andluminescent intensity of a molecule is determined from a plurality ofphotons that are emitted sequentially from the molecule, and theluminescent lifetime and luminescent intensity can be used to identifythe molecule.

Accordingly, in some aspects of the application, a reaction sample isexposed to a plurality of separate light pulses and a series of emittedphotons are detected and analyzed. In some embodiments, the series ofemitted photons provides information about a single molecule that ispresent and that does not change in the reaction sample over the time ofthe experiment. However, in some embodiments, the series of emittedphotons provides information about a series of different molecules thatare present at different times in the reaction sample (e.g., as areaction or process progresses).

Luminescent Labels

As used herein, a “luminescent label” is a molecule that absorbs one ormore photons and may subsequently emit one or more photons after one ormore time durations. In some embodiments, the term may be used togenerally refer to a non-reactant portion of a labeled reactant (e.g., aluminescent label can include a fluorophore and at least a portion of aspacer). In some embodiments, the term refers specifically to themolecule that absorbs and/or emits photons (e.g., the fluorophore). Insome embodiments, the term is used interchangeably with “luminescentmolecule” depending on context. In some embodiments, the luminescentlabel is a fluorophore (e.g., a “dye” or “fluorophore dye”, as usedherein interchangeably). In some embodiments, the luminescent label is arhodamine-based molecule. In some embodiments, the luminescent label isa cyanine-based molecule. In some embodiments, the luminescent label isa BODIPY®-based molecule.

Typically, the luminescent label comprises an aromatic or heteroaromaticcompound and can be a pyrene, anthracene, naphthalene, acridine,stilbene, indole, benzindole, oxazole, carbazole, thiazole,benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline,ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluoroscein, rhodamine or other like compound. Examples ofdyes include xanthene dyes, such as fluorescein or rhodamine dyes,including 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™), 6-carboxy-X-rhodamine(ROX). Examples of dyes also include naphthylamine dyes that have anamino group in the alpha or beta position. For example, naphthylaminocompounds include 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalenesulfonate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Other examples of dyes include coumarins, such as3-phenyl-7-isocyanatocoumarin; acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such asindodicarbocyanine 3 (Cy®3),(2Z)-2-[(E)-3-[3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfobenzo[e]indol-3-ium-2-yl]prop-2-enylidene]-3-ethyl-1,1-dimethyl-8-(trioxidanylsulfanyl)benzo[e]indole-6-sulfonate(Cy®3.5),2-[2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″]indolizino[8″,7″:5′,6′]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5-ium-14-sulfonate(Cy®3B), indodicarbocyanine 5 (Cy®5), indodicarbocyanine 5.5 (Cy®5.5),3-(-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin- 18-ium, 9-[2(or4)-[[[6-(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed®(5-chlorosulfonyl-2-(3-oxa-23-aza-9-azoniaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-16-yl)benzenesulfonate));BODIPY® dyes; benzoxazoles; stilbenes; pyrenes; and the like.

In certain embodiments, the luminescent label is a dye selected fromTable 3. The dyes listed in Table 3 are non-limiting, and theluminescent labels of the application may include dyes not listed inTable 3. In certain embodiments, the luminescent labels of one or moreluminescently labeled nucleotides is selected from Table 3. In certainembodiments, the luminescent labels of four or more luminescentlylabeled nucleotides is selected from Table 3.

TABLE 3 Examples of fluorophores Fluorophores 5/6-Carboxyrhodamine 6GChromis 678C DyLight ® 655-B1 5-Carboxyrhodamine 6G (9-[2- Chromis 678ZDyLight ® 655-B2 carboxy-4-[[(2,5-dioxo-1- pyrrolidinyl)oxy]carbonyl]phenyl]-3,6-bis(ethylamino)-2,7- dimethyl-xanthene) 6-Carboxyrhodamine6G (9-[5- Chromis 770A DyLight ® 655-B3 carboxy-2-(ethoxycarbonyl)phenyl]-3,6- bis(ethylamino)-2,7- dimethylxantheniumchloride) 6-TAMRA ™ (6- Chromis 770C DyLight ® 655-B4carboxytetramethylrhodamine) Alexa Fluor ® 350 (7-amino-3- Chromis 800ADyLight ® 662Q [2-(2,5-dioxopyrrolidin-1- yl)oxy-2-oxoethyl]-4-methyl-2-oxochromene-6-sulfonic acid) Alexa Fluor ® 405 (8-[2-[4-(2,5- Chromis800C DyLight ® 675-B1 dioxopyrrolidin-1- yl)oxycarbonylpiperidin-1-yl]-2-oxoethoxy]pyrene-1,3,6- trisulfonate) Alexa Fluor ® 430 ([9-[6-(2,5-Chromis 830A DyLight ® 675-B2 dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-8,8-dimethyl-2-oxo- 4-(trifluoromethyl)pyrano[3,2-g]quinolin-6-yl] methanesulfonate) Alexa Fluor ® 480 (3-(3-amino- Chromis830C DyLight ® 675-B3 6-imino-4,5- disulfonatoxanthen-9-yl)-4-carboxybenzoate) Alexa Fluor ® 488 (3-amino-6- Cy ® 3 DyLight ® 675-B4azaniumylidene-9-(2- (indodicarbocyanine 3) carboxyphenyl)xanthene-4,5-disulfonate) Alexa Fluor ® 514 (9- Cy ® 3.5 ((2Z)-2-[(E)-3- DyLight ®679-C5 azaniumylidene-6-[2-carboxy- [3-(5-carboxypentyl)-1,1-5-(2,5-dioxopyrrolidin-1- dimethyl-6,8- yl)oxycarbonylphenyl]-2,2,4-disulfobenzo[e]indol-3- trimethyl-12-sulfo-3,4- ium-2-yl]prop-2-dihydro-1H-chromeno[3,2-g] enylidene]-3-ethyl-1,1-quinoline-10-sulfonate) dimethyl-8- (trioxidanylsulfanyl)benzo[e]indole-6-sulfonate) Alexa Fluor ® 532 (5-(4- Cy ® 3B (2-{2-[(2,5-DyLight ® 680 {[(2,5-dioxopyrrolidin-1- dioxopyrrolidin-1-yl)oxy]-2-yl)oxy]carbonyl}phenyl)- oxoethyl}-16,16,18,18- 2,3,3,7,7,8-hexamethyl-tetramethyl- 2,3,7,8-tetrahydro-1H- 6,7,7a,8a,9,10,16,18-pyrano[3,2-f:5,6-f′]diindole- octahydrobenzo[2″,3″] 10,12-disulfonicacid) indolizino[8″,7″:5′,6′]pyrano[3′, 2′:3,4]pyrido[1,2-a]indol-5-ium-14-sulfonate) Alexa Fluor ® 546 (sodium 6-(2- Cy ® 5 DyLight ® 683Qcarboxy-3,4,6-trichloro-5-{[2- (indodicarbocyanine 5)({6-[(2,5-dioxopyrrolidin-1- yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]thio}phenyl)- 2,2,4,8,10,10-hexamethyl- 3,4,5a,8,9,10,11,12a-octahydro-2H-pyrano[3,2- g:5,6-g′]diquinolin-1-ium- 12,14-disulfonate)Alexa Fluor ® 555 (4-(3-amino- Dyomics ®-350 DyLight ® 690-B16-imino-4,5-disulfoxanthen-9- yl)benzene-1,3-dicarboxylic acid) AlexaFluor ® 568 ([13-[2- Dyomics ®-350XL DyLight ® 690-B2 carboxy-4-(2,5-dioxopyrrolidin-1- yl)oxycarbonylphenyl]- 7,7,19,19-tetramethyl-17-(sulfomethyl)-2-oxa-20-aza-6- azoniapentacyclo[12.8.0.0^(3,12).^(05,10).0^(16,21)]docosa- 1(14),3,5,8,10,12,15,17,21- nonaen-9-yl]methanesulfonate) Alexa Fluor ® 594 ([13-[2- Dyomics ®-360XLDyLight ® 696Q carboxy-4-(2,5- dioxopyrrolidin-1- yl)oxycarbonylphenyl]-6,7,7,19,19,20-hexamethyl-17- (sulfomethyl)-2-oxa-20-aza-6-azoniapentacyclo[12.8.0.0^(3,12). ^(05,10).0^(16,21)]docosa-1(14),3,5,8,10,12,15,17,21- nonaen-9-yl] methanesulfonate) Alexa Fluor ®610-X (2,3,5- Dyomics ®-370XL DyLight ® 700-B1 trichloro-4-[2-[[6-(2,5-dioxopyrrolidin-1-yl)oxy-6- oxohexyl]amino]-2- oxoethyl]sulfanyl-6-[6,7,7,19,19,20-hexamethyl- 9,17-bis(sulfonatomethyl)-2- oxa-20-aza-6-azoniapentacyclo[12.8.0.0^(3,12). ^(05,10).0^(16,21)]docosa-1(14),3,5,8,10,12,15,17,21- nonaen-13- yl]benzoate;triethylazanium)Alexa Fluor ® 633 Dyomics ®-375XL DyLight ® 700-B1 Alexa Fluor ® 647(2-[5-[3,3- Dyomics ®-380XL DyLight ® 730-B1 dimethyl-5-sulfo-1-(3-sulfopropyl)indol-1-ium-2- yl]penta-2,4-dienylidene]-3-methyl-3-[5-oxo-5-(6- phosphonooxyhexylamino)pentyl]-1-(3-sulfopropyl)indole-5- sulfonic acid) Alexa Fluor ® 660Dyomics ®-390XL DyLight ® 730-B2 Alexa Fluor ® 680 Dyomics ®-405DyLight ® 730-B3 Alexa Fluor ® 700 Dyomics ®-415 DyLight ® 730-B4 AlexaFluor ® 750 Dyomics ®-430 DyLight ® 747 Alexa Fluor ® 790 Dyomics ®-431DyLight ® 747-B1 AMCA (aminomethylcoumarin Dyomics ®-478 DyLight ®747-B2 acetate) ATTO ™ 390 (4-(4,6,8,8- Dyomics ®-480XL DyLight ® 747-B3tetramethyl-2-oxo-6,7- dihydropyrano[3, 2-g]quinolin- 9-yl)butanoicacid) ATTO ™ 425 (4-(3- Dyomics ®-481XL DyLight ® 747-B4ethoxycarbonyl-6,8,8- trimethyl-2-oxo-6,7- dihydropyrano[3,2-g]quinolin-9-yl)butanoic acid) ATTO ™ 465 (4-(3-amino-6- Dyomics ®-485XL DyLight ®755 iminoacridin-10-yl)butanoic acid) ATTO ™ 488 ([6-amino-9-[2-(3-Dyomics ®-490 DyLight ® 766Q carboxypropylcarbamoyl)phenyl]-4,5-disulfoxanthen-3- ylidene]azanium) ATTO ™ 495 (4-[3,6-Dyomics ®-495 DyLight ® 775-B2 bis(dimethylamino)acridin-10-ium-10-yl]butanoic acid) ATTO ™ 514 Dyomics ®-505 DyLight ® 775-B3ATTO ™ 520 ([9-(2- Dyomics ®-510XL DyLight ® 775-B4carboxyethyl)-6-(ethylamino)- 2,7-dimethylxanthen-3-ylidene]-ethylazanium) ATTO ™ 532 Dyomics ®-511XL DyLight ® 780-B1ATTO ™ 542 Dyomics ®-520XL DyLight ® 780-B2 ATTO ™ 550 Dyomics ®-521XLDyLight ® 780-B3 ATTO ™ 565 (2-(6,20-diethyl-2- Dyomics ®-530 DyLight ®800 oxa-20-aza-6- azoniapentacyclo[12.8.0.0^(3,12).^(05,10).0^(16,21)]docosa- 1(14)3,5,10,12,15,21-heptaen-13-yl)terephthalic acid) ATTO ™ 590 (4-(6,20-diethyl-Dyomics ®-547 DyLight ® 830-B2 7,7,9,17,19,19-hexamethyl-2-oxa-20-aza-6- azoniapentacyclo[12.8.0.0^(3,12).^(05,10).0^(16,21)]docosa- 1(14)3,5,8,10,12,15,17,21-nonaen-13-yl)benzene-1,3- dicarboxylic acid) ATTO ™ 610 (4-[9-Dyomics ®-547P1 eFluor ® 450 (dimethylamino)-11,11-dimethyl-3,4-dihydro-2H- naphtho[2,3-g]quinolin-1-ium- 1-yl]butanoicacid) ATTO ™ 620 (N-(10-(2-((3- Dyomics ®-548 Eosin(2-(2,4,5,7-tetrabromo-3- carboxypropyl)(methyl) oxido-6-oxoxanthen-9-carbamoyl)phenyl)-7- yl)benzoate) (dimethylamino)-9,9-dimethylanthracen-2(9H)- ylidene)-N- methylmethanaminium) ATTO ™ 633Dyomics ®-549 FITC (3′,6′-dihydroxy-6- isothiocyanatospiro[2-benzofuran-3,9′-xanthene]-1- one) ATTO ™ 647 (11-(3- Dyomics ®-549P1Fluorescein (2-(6-hydroxy-3- carboxypropyl)-1-ethyl-oxo-3H-xanthen-9-yl)benzoic 2,2,8,10,10,13,13- acid)heptamethyl-4-(sulfomethyl)- 2,8,9,10,11,13-hexahydrobenzo[1,2-g:5,4-g′] diquinolin-1-ium) ATTO ™ 647N ((2-(7-Ethyl-Dyomics ®-550 HiLyte ™ Fluor 405 3,3,8,8,10-pentamethyl-7-aza- 21-azoniahexacyclo[15.7.1.0^(2,15). ^(04,13).0^(6,11).0^(21,25)]pentacosa-1,4(13),5,11,14,16,18,21(25)- octaen-14-yl)-N-methyl-N-(4-oxopentyl)benzamide) ATTO ™ 655 ([6-(3- Dyomics ®-554 HiLyte ™ Fluor 488carboxypropyl)-20-ethyl-7,7- dimethyl-2-oxa-6,13-diaza-20-azoniapentacyclo[12.8.0.0^(3.12). ^(05.10).0^(16.21)]docosa-1(22),3(12),4,10,13,15,20- heptaen-9-yl] methanesulfonate) ATTO ™ 665Dyomics ®-555 HiLyte ™ Fluor 532 ATTO ™ 680 ([6-(3- Dyomics ®-556HiLyte ™ Fluor 555 carboxypropyl)-20-ethyl-7,7-dimethyl-2-oxa-6,13-diaza-20- azoniapentacyclo[12.8.0.^(03,12).^(05,10).0^(16,21)]docosa- 1(22),3(12),4,8,10,13,15,20- octaen-9-yl]methanesulfonate) ATTO ™ 700 ((11-(3- Dyomics ®-560 HiLyte ™ Fluor 594carboxypropyl)-1-ethyl- 2,2,4,10,10-pentamethyl- 10,11-dihydro-2H-dipyrido[3,2-b:2′,3′-i] phenoxazin-1-ium-8-yl) methanesulfonate) ATTO ™725 Dyomics ®-590 HiLyte™ Fluor 647 ATTO ™ 740 Dyomics ®-591 HiLyte™Fluor 680 ATTO ™ Oxa12 Dyomics ®-594 HiLyte™ Fluor 750 ATTO ™ Rho101Dyomics ®-601XL IRDye ® 680LT (sodium 2- ((1E,3Z,5Z)-3-(3-(4-carboxybutyl)phenyl)-5-(1,1- dimethyl-6,8-disulfonato-3-(3-sulfonatopropyl)-1,3-dihydro- 2H-benzo[e]indol-2-ylidene)penta-1,3-dien-1-yl)- 1,1-dimethyl-3-(3- sulfonatopropyl)-1H-benzo[e]indol-3-ium-6,8- disulfonate) ATTO ™ Rho11 (6-(2-((3-Dyomics ®-605 IRDye ® 750 (tetrasodium carboxypropyl)(methyl)carbamoyl)mono(2-((E)-2-((E)-3′-(4- phenyl)-1,11-diethyl-carboxybutyl)-6-(2-((E)-3,3- 3,4,8,9,10,11-hexahydro-2H-dimethyl-5-sulfonato-1-(4- pyrano[3,2-g:5,6-g′]diquinolin-sulfonatobutyl)indolin-2- 1-ium) ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2- yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1- ium-5-sulfonate) ATTO ™ Rho12 (6-(2-((3-Dyomics ®-610 IRDye ® 800CW (tetrasodium carboxypropyl)(methyl)carbamono(2-((E)-2-((E)-3-(2-((E)-1- moyl)phenyl)-1,11-diethyl-(5-carboxypentyl)-3,3- 2,2,4,8,10,10-hexamethyl-dimethyl-5-sulfonatoindolin-2- 3,4,8,9,10,11-hexahydro-2H-ylidene)ethylidene)-2-(4- pyrano[3,2-g:5,6-g′]diquinolin-sulfonatophenoxy)cyclohex-1- 1-ium) en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1- ium-5-sulfonate) ATTO ™ Rho13 (6-(2-((3-Dyomics ®-615 JOE carboxypropyl)(methyl)carbamoyl) phenyl)-1,11-diethyl-2,2,4,8,10,10-hexamethyl- 10,11-dihydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium) ATTO ™ Rho14 (6-(2-((3- Dyomics ®-630LightCycler ® 640R carboxypropyl)(methyl) carbamoyl)-3,4,5,6-tetrachlorophenyl)-1,11- diethyl-2,2,4,8,10,10-hexamethyl-10,11-dihydro-2H- pyrano[3,2-g:5,6-g′]diquinolin- 1-ium)ATTO ™ Rho3B (N-(13-(2-((3- Dyomics ®-631 LightCycler ® Red 610carboxypropyl)(methyl) carbamoyl)phenyl)-9- (diethylamino)-8,9-dihydro-3H-dibenzo[b,i]xanthen-3- ylidene)-N-ethylethanaminium ATTO ™ Rho6G(N-(9-(2-((3- Dyomics ®-632 LightCycler ® Red 640carboxypropyl)(methyl)carbamoyl) phenyl)-6-(ethylamino)-2,7-dimethyl-3H-xanthen-3- ylidene)-N- methylmethanaminium) ATTO ™Thio12 ([9-[2-[3- Dyomics ®-633 LightCycler ® Red 670carboxypropyl(methyl)carbamoyl] phenyl]-6- (dimethylamino)thioxanthen-3-ylidene]-dimethylazanium) BD Horizon ™ V450 Dyomics ®-634LightCycler ® Red 705 BODIPY ® 493/503 (3-(5,5- Dyomics ®-635 LissamineRhodamine B (2-[3- difluoro-1,3,7,9-tetramethyl- (diethylamino)-6-3H,5H-5λ4-dipyrrolo[1,2- diethylazaniumylidenexanthen-c:2′,1′-f][1,3,2]diazaborinin- 9-yl]-5-sulfobenzenesulfonate)10-yl)propanoic acid) BODIPY ® 530/550 (3-(5,5- Dyomics ®-636Napthofluorescein (7′,19′- difluoro-1,3-diphenyl-3H,5H-dihydroxyspiro[2-benzofuran- 5λ4-dipyrrolo[1,2-c:2′,1′- 3,13′-2-f][1,3,2]diazaborinin-7- oxapentacyclo[12.8.0.0³¹². yl)propanoic acid)0^(4,9).^(017,22)]docosa- 1(14),3(12),4(9),5,7,10,15,17(22),18,20-decaene]-1-one) BODIPY ® 558/568 (3-(5,5- Dyomics ®-647 OregonGreen ® 488 (4-(2,7- difluoro-3-(thiophen-2-yl)- difluoro-3-hydroxy-6-3H,5H-5λ4-dipyrrolo[1,2- oxoxanthen-9-yl)benzene-l,3-c:2′,1′-f][1,3,2]diazaborinin-7- dicarboxylic acid) yl)propanoic acid)BODIPY ® 564/570 ((E)-3-(5,5- Dyomics ®-647P1 Oregon Green ® 514difluoro-3-styryl-3H,5H-5λ4- dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-7- yl)propanoic acid) BODIPY ® 576/589 (3-(5,5-Dyomics ®-648 Pacific Blue ™ difluoro-3-(1H-pyrrol-2-yl)-3H,5H-5λ4-dipyrrolo[1,2- c:2′,1′-f][1,3,2]diazaborinin-7- yl)propanoicacid) BODIPY ® 581/591 (3-(5,5- Dyomics ®-648P1 Pacific Green ™difluoro-3-((1E,3E)-4- phenylbuta-1,3-dien-1-yl)-3H,5H-5λ4-dipyrrolo[1,2- c:2′,1′-f][1,3,2]diazaborinin-7- yl)propanoicacid) BODIPY ® 630/650 ((E)-6-(2-(4- Dyomics ®-649 Pacific Orange ™(2-(5,5-difluoro-3-(thiophen-2- yl)-3H,5H-5λ4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-7- yl)vinyl)phenoxy)acetamido) hexanoicacid) BODIPY ® 650/665 ((E)-6-(2-(4- Dyomics ®-649P1 PET(2-(5,5-difluoro-3-(1H-pyrrol- 2-yl)-3H,5H-5λ4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-7- yl)vinyl)phenoxy)acetamido)he xanoicacid) BODIPY ® FL (3-(5,5-difluoro- Dyomics ®-650 PF3501,3-dimethyl-3H,5H-54- dipyrrolo[1,2-c:2′,1′- f][1,3,2]diazaborinin-7-yl)propanoic acid) BODIPY ® FL-X (6-(3-(5,5- Dyomics ®-651 PF405difluoro-1,3-dimethyl-3H,5H- 5λ4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-7- yl)propanamido)hexanoic acid) BODIPY ® R6G(3-(5,5-difluoro- Dyomics ®-652 PF415 3-phenyl-3H,5H-5λ4-dipyrrolo[1,2-c:2′,1′- f][1,3,2]diazaborinin-7- yl)propanoic acid)BODIPY ® TMR (6-(3-(5,5- Dyomics ®-654 PF488difluoro-3-(4-methoxyphenyl)- 7,9-dimethyl-3H,5H-5λ4-dipyrrolo[1,2-c:2′,1′- f][1,3,2]diazaborinin-8- yl)propanamido)hexanoicacid) BODIPY ® TR (7-(4-(2-((5- Dyomics ®-675 PF505carboxypentyl)amino)-2- oxoethoxy)phenyl)-5,5-difluoro-3-(thiophen-2-yl)-5H- 5λ4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium) C5.5 (2,5-dioxopyrrolidin-1-ylDyomics ®-676 PF532 6-(1,1-dimethyl-2-((1E,3E,5E)-5-(1,1,3-trimethyl-1,3-dihydro- 2H-benzo[e]indol-2-ylidene)penta-1,3-dien-1-yl)- 1H-3λ4-benzo[e]indol-3- yl)hexanoate) C7(2,5-dioxopyrrolidin-1-yl 6- Dyomics ®-677 PF546(3,3-dimethyl-2-((E)-2-((E)-3- (2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex- 1-en-1-yl)vinyl)-3H-1λ4-indol-1-yl)hexanoate) CAL Fluor ® Gold 540 (4,5- Dyomics ®-678 PF555Pdichloro-2-(2,7-dichloro-6- hydroxy-3-oxo-3H-xanthen-9- yl)benzoic acid)CAL Fluor ® Green 510 (2-(6- Dyomics ®-679P1 PF568hydroxy-3-oxo-3H-xanthen-9- yl)benzoic acid) CAL Fluor ® Orange 560((E)-N- Dyomics ®-680 PF594 (9-(2-carboxyphenyl)-6-(ethylamino)-2,7-dimethyl-3H- xanthen-3- ylidene)ethanaminium) CALFluor ® Red 590 (N-(6- Dyomics ®-681 PF610 (diethylamino)-9-(2-(1-(methyl(propyl)amino)vinyl) phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium) CAL Fluor ® Red 610 Dyomics ®-682 PF633P CALFluor ® Red 615 Dyomics ®-700 PF647P CAL Fluor ® Red 635 (6-(2-Dyomics ®-701 Quasar ® 570 (2-((1E,3E)-3-(3-carboxy-4,5-dimethylphenyl)- (5-carboxypentyl)-1,1-1,11-diethyl-2,2,4,8,10,10- dimethyl-1,3-dihydro-2H-hexamethyl-10,11-dihydro-2H- benzo[e]indol-2-ylidene)prop-pyrano[3,2-g:5,6-g′]diquinolin- 1-en-1-yl)-3-ethyl-1,1-dimethyl- 1-ium)1H-benzo[e]indol-3-ium) Cascade ® Blue (8-hydroxy-1,8- Dyomics ®-703Quasar ® 670 (2-((1E,3E)-5-((E)- dihydropyrene-1,3,6-1-(5-carboxypentyl)-3,3- trisulfonate) dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1- ethyl-3,3-dimethyl-3H-indol-1- ium) CF ™350 Dyomics ®-704 Quasar ® 705 (2-((1E,3E,5E)-5-(3-(5-carboxypentyl)-1,1- dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)penta- 1,3-dien-1-yl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol-3- ium) CF ™ 405M Dyomics ®-730 Rhoadmine 123([6-amino-9-(2- methoxycarbonylphenyl)xanthen- 3-ylidene]azanium) CF ™405S Dyomics ®-731 Rhodamine 6G ([9-(2- ethoxycarbonylphenyl)-6-(ethylamino)-2,7- dimethylxanthen-3-ylidene]- ethylazanium) CF ™ 488ADyomics ®-732 Rhodamine B ([9-(2- carboxyphenyl)-6-(diethylamino)xanthen-3- ylidene]-diethylazanium) CF ™ 514 Dyomics ®-734Rhodamine Green ([6-amino-9- (2,5-dicarboxyphenyl)xanthen-3-ylidene]azanium) CF ™ 532 Dyomics ®-749 Rhodamine Green-X (2-(6-amino-3-(l4-azaneylidene)-3H- xanthen-9-yl)benzoic acid) CF ™ 543Dyomics ®-749P1 Rhodamine Red (4-(3-((2I5- butan-2-yl)imino)-6-(diethylamino)-3H-xanthen-9- yl)benzene-1,3-disulfonate) CF ™ 546Dyomics ®-750 ROX CF ™ 555 Dyomics ®-751 ROX CF ™ 568 Dyomics ®-752Seta ™ 375 CF ™ 594 Dyomics ®-754 Seta ™ 470 CF ™ 620R Dyomics ®-776Seta ™ 555 CF ™ 633 Dyomics ®-777 Seta ™ 632 CF ™ 633-V1 Dyomics ®-778Seta ™ 633 CF ™ 640R Dyomics ®-780 Seta ™ 650 CF ™ 640R-Vl Dyomics ®-781Seta ™ 660 CF ™ 640R-V2 Dyomics ®-782 Seta ™ 670 CF ™ 660C Dyomics ®-800Seta ™ 680 CF ™ 660R Dyomics ®-831 Seta ™ 700 CF ™ 680 DyLight ® 350Seta ™ 750 CF ™ 680R DyLight ® 405 Seta ™ 780 CF ™ 680R-V1 DyLight ®415-Co1 Seta ™ APC-780 CF ™ 750 DyLight ® 425Q Seta ™ PerCP-680 CF ™ 770DyLight ® 485-LS Seta ™ R-PE-670 CF ™ 790 DyLight ® 488 Seta ™ 646Chromeo ™ 642 DyLight ® 504Q Seta ™ u 380 Chromis 425N DyLight ® 510-LSSeta ™ u 425 Chromis 500N DyLight ® 515-LS Seta ™ u 647 Chromis 515NDyLight ® 521-LS Seta ™ u 405 Chromis 530N DyLight ® 530-R2Sulforhodamine 101 (2-(3-oxa- 23-aza-9- azoniaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(23,28)]octacosa-1(27),2(17),4,9(28),13,15,18- heptaen-16-yl)-5- sulfobenzenesulfonate)Chromis 550A DyLight ® 543Q TAMRA ™ (6- carboxytetramethylrhodamine)Chromis 550C DyLight ® 550 TET Chromis 550Z DyLight ® 554-R0 Texas Red ®(5-chlorosulfonyl-2- (3-oxa-23-aza-9-azoniaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa- 1(27),2(17),4,9(28),13,15,18-heptaen-16- yl)benzenesulfonate) Chromis 560N DyLight ® 554-R1 TMR(5-carboxy-2-(6- (dimethylamino)-3- (dimethyliminio)-2,3-dihydro-1H-xanthen-9-yl)benzoate) Chromis 570N DyLight ® 590-R2 TRITC(2-(6-(dimethylamino)-3- (dimethyliminio)-2,3-dihydro-1H-xanthen-9-yl)-4- thiocyanatobenzoate) Chromis 577N DyLight ® 594Yakima Yellow ™ (2-(7-(2- carboxylatoethyl)-2,5-dichloro-6-hydroxy-4-methyl-3-oxo-3H- xanthen-9-yl)-4,5- dichlorobenzoate)Chromis 600N DyLight ® 610-B1 Zenon ® Chromis 630N DyLight ® 615-B2 Zy3(1-(5-carboxypentyl)-2-((E)- 3-((E)-1-ethyl-3,3-dimethyl-5-sulfoindolin-2-ylidene)prop-1- en-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) Chromis 645A DyLight ® 633 Zy5(1-(5-carboxypentyl)-2- ((1E,3E)-5-((E)-1-ethyl-3,3-dimethyl-5-sulfoindolin-2- ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5- sulfonate) Chromis 645C DyLight ® 633-B1Zy5.5 (3-(5-carboxypentyl)-2- ((1E,3E,5E)-5-(3-ethyl-1,1-dimethyl-6,8-disulfo-1,3- dihydro-2H-benzo[e]indol-2-ylidene)penta-1,3-dien-1-yl)- 1,1-dimethyl-8-sulfo-1H-benzo[e]indol-3-ium-6- sulfonate) Chromis 645Z DyLight ® 633-B2 Zy7(1-(5-carboxypentyl)-2- ((1E,3E,5E)-7-((E)-1-ethyl-3,3-dimethylindolin-2- ylidene)hepta-1,3,5-trien-1-yl)-3,3-dimethyl-3H-indol-1-ium) Chromis 678A DyLight ® 650 Abberior ® Star635 Square 635 Square 650 Square 660 Square 672 Square 680 Abberior ®Star 440SXP Abberior ® Star470SXP Abberior ® Star 488 Abberior ® Star512 Abberior ® Star 520SXP Abberior ® Star 580 Abberior ® Star 600Abberior ® Star 635 Abberior ® Star 635P Abberior ® Star RED

Dyes may also be classified based on the wavelength of maximumabsorbance or emitted luminescence. Table 4 provides exemplaryfluorophores grouped into columns according to approximate wavelength ofmaximum absorbance. The dyes listed in Table 4 are non-limiting, and theluminescent labels of the application may include dyes not listed inTable 4. The exact maximum absorbance or emission wavelength may notcorrespond to the indicated spectral ranges. In certain, embodiments,the luminescent labels of one or more luminescently labeled nucleotidesis selected from the “Red” group listed in Table 4. In certainembodiments, the luminescent labels of one or more luminescently labelednucleotides is selected from the “Green” group listed in Table 4. Incertain embodiments, the luminescent labels of one or more luminescentlylabeled nucleotides is selected from the “Yellow/Orange” group listed inTable 4. In certain embodiments, the luminescent labels of fournucleotides are selected such that all are selected from one of the“Red”, “Yellow/Orange”, or “Green” group listed in Table 4. In certainembodiments, the luminescent labels of four nucleotides are selectedsuch that three are selected from a first group of the “Red”,“Yellow/Orange”, and “Green” groups listed in Table 4, and the fourth isselected from a second group of the “Red”, “Yellow/Orange”, and “Green”groups listed in Table 4. In certain embodiments, the luminescent labelsof four nucleotides are selected such that two are selected from a firstof the “Red”, “Yellow/Orange”, and “Green” group listed in Table 4, andthe third and fourth are selected from a second group of the “Red”,“Yellow/Orange”, and “Green” groups listed in Table 4. In certainembodiments, the luminescent labels of four nucleotides are selectedsuch that two are selected from a first of the “Red”, “Yellow/Orange”,and “Green” groups listed in Table 4, and a third is selected from asecond group of the “Red”, “Yellow/Orange”, and “Green” groups listed inTable 4, and a fourth is selected from a third group of the “Red”,“Yellow/Orange”, and “Green” groups listed in Table 4.

TABLE 4 Examples of fluorophores by spectral range “Green” 520-570 nm“Yellow/Orange” 570-620 nm “Red” 620-670 nm 5/6-Carboxyrhodamine 6GAlexa Fluor ® 594 ([13-[2- Alexa Fluor ® 633carboxy-4-(2,5-dioxopyrrolidin- 1-yl)oxycarbonylphenyl]-6,7,7,19,19,20-hexamethyl-17- (sulfomethyl)-2-oxa-20-aza-6-azoniapentacyclo[12.8.0.0^(3,12). 0^(5,10).0^(16,21)]docosa-1(14),3,5,8,10,12,15,17,21- nonaen-9-yl]methanesulfonate) 6-TAMRA ™ (6-Alexa Fluor ® 610-X (2,3,5- Alexa Fluor ® 647 (2-[5-[3,3-carboxytetramethylrhodamine) trichloro-4-[2-[[6-(2,5-dimethyl-5-sulfo-1-(3- dioxopyrrolidin-1-yl)oxy-6-sulfopropyl)indol-1-ium-2- oxohexyl]amino]-2-yl]penta-2,4-dienylidene]-3- oxoethyl]sulfanyl-6- methyl-3-[5-oxo-5-(6-[6,7,7,19,19,20-hexamethyl- phosphonooxyhexylamino)9,17-bis(sulfonatomethyl)-2- pentyl]-1-(3-sulfopropyl) oxa-20-aza-6-indole-5-sulfonic acid) azoniapentacyclo[12.8.0.0^(3,12).0^(5,10).0^(16,21)]docosa- 1(14),3,5,8,10,12,15,17,21- nonaen-13-yl]benzoate;triethylazanium) Alexa Fluor ® 532 (5-(4-{[(2,5- ATTO ™ 590(4-(6,20-diethyl- Alexa Fluor ® 660 dioxopyrrolidin-1-7,7,9,17,19,19-hexamethyl-2- yl)oxy]carbonyl}phenyl)- oxa-20-aza-6-2,3,3,7,7,8-hexamethyl- azoniapentacyclo[12.8.0.0^(3,12).2,3,7,8-tetrahydro-1H- 0^(5,10).0^(16,21)]docosa-pyrano[3,2-f:5,6-f′]diindole- 1(14),3,5,8,10,12,15,17,21-10,12-disulfonic acid) nonaen-13-yl)benzene-1,3- dicarboxylic acid)Alexa Fluor ® 546 (sodium 6- ATTO ™ 610 (4-[9- ATTO ™ 633(2-carboxy-3,4,6-trichloro-5- (dimethylamino)-11,11-{[2-({6-[(2,5-dioxopyrrolidin- dimethyl-3,4-dihydro-2H-l-yl)oxy]-6-oxohexyl}amino)- naphtho[2,3-g]quinolin-1-ium-1-2-oxoethyl]thio}phenyl)- yl]butanoic acid) 2,2,4,8,10,10-hexamethyl-3,4,5a,8,9,10,11,12a- octahydro-2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium- 12,14-disulfonate) Alexa Fluor ® 555 (4-(3-ATTO ™ 620 (N-(10-(2-((3- ATTO ™ 647 (11-(3- amino-6-imino-4,5-carboxypropyl)(methyl)carbamo carboxypropyl)-1-ethyl- disulfoxanthen-9-yl)phenyl)-7-(dimethylamino)- 2,2,8,10,10,13,13-yl)benzene-1,3-dicarboxylic 9,9-dimethylanthracen-2(9H)-heptamethyl-4-(sulfomethyl)- acid) ylidene)-N- 2,8,9,10,11,13-methylmethanaminium) hexahydrobenzo[1,2-g:5,4- g′]diquinolin-1-ium)Alexa Fluor ® 568 ([13-[2- BODIPY ® 576/589 3-(5,5- ATTO ™ 647N((2-(7-Ethyl- carboxy-4-(2,5- difluoro-3-(1H-pyrrol-2-yl)-3,3,8,8,10-pentamethyl-7- dioxopyrrolidin-1-3H,5H-5λ4-dipyrrolo[1,2-c:2′,1′- aza-21- yl)oxycarbonylphenyl]-f][1,3,2]diazaborinin-7- azoniahexacyclo[15.7.1.0^(2,15).7,7,19,19-tetramethyl-17- yl)propanoic acid) 04^(,13).06^(,11).0^(21,25)pentacosa- (sulfomethyl)-2-oxa-20-aza- 1,4(13),5,11,14,16,18,21(25)- 6-octaen-14-yl)-N-methyl-N-(4- azoniapentacyclo[12.8.0.03,oxopentyl)benzamide) 12.05,10.016,21]docosa- 1(14),3,5,8,10,12,15,17,21-nonaen-9- yl]methanesulfonate) ATTO ™ 520 ([9-(2- BODIPY ® 581/591(3-(5,5- ATTO ™ 655 ([6-(3- carboxyethyl)-6- difluoro-3-((1E,3E)-4-carboxypropyl)-20-ethyl-7,7- (ethylamino)-2,7-phenylbuta-1,3-dien-1-yl)- dimethyl-2-oxa-6,13-diaza-dimethylxanthen-3-ylidene]- 3H,5H-5λ4-dipyrrolo[l,2-c:2′,1′- 20-ethylazanium) f][1,3,2]diazaborinin-7- azoniapentacyclo[12.8.0.0^(3,12).yl)propanoic acid) 0^(5,10).0^(16,21)]docosa- 1(22),3(12),4,10,13,15,20-heptaen-9- yl]methanesulfonate ATTO ™ 532 CF ™ 594 ATTO ™ 665 ATTO ™ 542CF ™ 620R ATTO ™ 680 ([6-(3- carboxypropyl)-20-ethyl-7,7-dimethyl-2-oxa-6,13-diaza- 20- azoniapentacyclo[12.8.0.0^(3,12).0^(5,10).0^(16,21)]docosa- 1(22),3(12),4,8,10,13,15,20- octaen-9-yl]methanesulfonate) ATTO ™ 550 Chromis 570N ATTO ™ Rho14 (6-(2-((3-carboxypropyl)(methyl) carbamoyl)-3,4,5,6- tetrachlorophenyl)-1,11-diethyl-2,2,4,8,10,10- hexamethyl-10,11-dihydro- 2H-pyrano[3,2-g:5,6-g′]diquinolin-1-ium) ATTO ™ 565 (2-(6,20-diethyl- Chromis 577N BODIPY ®630/650 ((E)-6-(2- 2-oxa-20-aza-6- (4-(2-(5,5-difluoro-3-azoniapentacyclo[12.8.0.^(03,12). (thiophen-2-yl)-3H,5H-5λ4-0^(5,10).0^(16,21)]docosa- dipyrrolo[1,2-c:2′,1′- 1(14),3,5,10,12,15,21-f][1,3,2]diazaborinin-7- heptaen-13-yl)terephthalicyl)vinyl)phenoxy)acetamido) acid) hexanoic acid) BODIPY ® 530/550(3-(5,5- Chromis 600N BODIPY ® 650/665 ((E)-6-(2-difluoro-l,3-diphenyl-3H,5H- (4-(2-(5,5-difluoro-3-(1H-5λ4-dipyrrolo[1,2-c:2′,1′- pyrrol-2-yl)-3H,5H-5λ4-f][1,3,2]diazaborinin-7- dipyrrolo[1,2-c:2′,1′- yl)propanoic acid)f][1,3,2]diazaborinin-7- yl)vinyl)phenoxy)acetamido) hexanoic acid)BODIPY ® 558/568 (3-(5,5- Dyomics ®-590 CAL Fluor ® Red 635 (6-(2-difluoro-3-(thiophen-2-yl)- carboxy-4,5-dimethylphenyl)-3H,5H-5λ4-dipyrrolo[1,2- 1,11-diethyl-2,2,4,8,10,10-c:2′,1′-f][l,3,2]diazaborinin- hexamethyl-10,11-dihydro- 7-yl)propanoicacid) 2H-pyrano[3,2-g:5,6- g′]diquinolin-1-ium) BODIPY ® 564/570 ((E)-3-Dyomics ®-591 CF ™ 633-V1 (5,5-difluoro-3-styryl-3H,5H-5λ4-dipyrrolo[1,2-c:2′,1′- f][1,3,2]diazaborinin-7- yl)propanoic acid)CF ™ 514 Dyomics ®-594 CF ™ 640R-V1 CF ™ 532 Dyomics ®-601XL CF ™ 633CF ™ 543 Dyomics ®-605 CF ™ 640R CF ™ 546 Dyomics ®-610 CF ™ 640R-V2CF ™ 555 Dyomics ®-615 CF ™ 660C CF568 DyLight ® 590-R2 CF ™ 660RChromis 530N DyLight ® 594 CF ™ 680 Chromis 550A DyLight ® 610-B1 CF ™680R Chromis 550C DyLight ® 615-B2 CF ™ 680R-V1 Chromis 550Z HiLyte ™Fluor 594 Chromeo ™ 642 Chromis 560N LightCycler ® Red 610 Chromis 630NCy ® 3 (indodicarbocyanine 3) PF594 Chromis 645A Cy ® 3.5((2Z)-2-[(E)-3-[3-(5- PF594 Chromis 645A carboxypentyl)-1,1-dimethyl-6,8- disulfobenzo[e]indol-3-ium- 2-yl]prop-2-enylidene]-3-ethyl-1,1-dimethyl-8- (trioxidanylsulfanyl)benzo[e] indole-6-sulfonate)Cy ® 3B (2-{2-[(2,5- PF610 Chromis 645C dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-16,16,18,18- tetramethyl- 6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″]indolizino [8″,7″:5′,6′]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5- ium-14-sulfonate) Dyomics ®-530 Quasar ® 570(2-((1E,3E)-3-(3-(5- Chromis 645Z carboxypentyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2- ylidene)prop-1-en-1-yl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol- 3-ium) Dyomics ® -547 Abberior ® Star 580Cy ® 5 (indodicarbocyanine 5) Dyomics-547P1 Abberior ® Star 600 Cy ® 5.5(indodicarbocyanine 5.5) Dyomics ®-548 Dyomics ®-630 Dyomics ®-549P1Dyomics ®-631 Dyomics ®-550 Dyomics ®-632 Dyomics ®-554 Dyomics ®-633Dyomics ®-555 Dyomics ®-634 Dyomics ®-556 Dyomics ®-635 Dyomics ®-560Dyomics ®-636 DyLight ® 521-LS Dyomics ®-647 DyLight ® 530-R2Dyomics ®-647P1 DyLight ® 543Q Dyomics ®-648 DyLight ® 550Dyomics ®-648P1 DyLight ® 554-R0 Dyomics ®-649 DyLight ® 554-R1Dyomics ®-649P1 HiLyte ™ Fluor 532 Dyomics ®-650 HiLyte ™ Fluor 555Dyomics ®-651 PF532 Dyomics ®-652 PF546 Dyomics ®-654 PF555P DyLight ®633 PF568 DyLight ® 633-B1 Seta ™ 555 DyLight ® 633-B2 Abberior ® Star520SXP DyLight ® 650 DyLight ® 655-B1 DyLight ® 655-B2 DyLight ® 655-B3DyLight ® 655-B4 DyLight ® 662Q DyLight ® 680 DyLight ® 683Q HiLyte ™Fluor 647 HiLyte ™ Fluor 680 LightCycler ® 640R LightCycler ® Red 640LightCycler ® Red 670 PF633P PF647P Quasar ® 670 (2-((1E,3E)-5-((E)-1-(5-carboxypentyl)-3,3- dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)- 1-ethyl-3,3-dimethyl-3H- indol-1-ium)Seta ™ 632 Seta ™ 633 Seta ™ 650 Seta ™ 660 Seta ™ 670 Seta ™ Tau 647Square 635 Square 650 Square 660 Abberior ® Star 635 Abberior ® Star635P Abberior ® Star RED

In certain embodiments, the luminescent label may comprise a first andsecond chromophore. In some embodiments, an excited state of the firstchromophore is capable of relaxation via an energy transfer to thesecond chromophore. In some embodiments, the energy transfer is aFörster resonance energy transfer (FRET). Such a FRET pair may be usefulfor providing a luminescent label with properties that make the labeleasier to differentiate from amongst a plurality of luminescent labels.In certain embodiments, the FRET pair may absorb excitation energy in afirst spectral range and emit luminescence in a second spectral range.

In some embodiments, the luminescent label is attached within a linkermolecule described herein (e.g., integrated into an oligomeric orpolymeric linker). Table 5 provides several examples of fluorophorescompatible with such conjugation strategy in the context of anoligonucleotide strand. As shown, the cyanine-based fluorophores inTable 5 are conjugated to a 3′ end of a first strand portion and a 5′end of a second strand portion such that the fluorophore itself formspart of the covalent linkage within the oligonucleotide strand (e.g.,the luminescent label is attached without the use of a spacer). Itshould be appreciated that, in some embodiments, these and similar typesof fluorophores can be attached within any class of oligomeric orpolymeric structure. For example, in some embodiments, a fluorophore isattached within a peptide via conjugation to a C-terminal end of a firstpeptide portion and an N-terminal end of a second peptide portion. Insome embodiments, a single linker molecule comprises two or morefluorophores attached within the linker (e.g., two, three, four, five,six, or more than six dyes attached within a single linker). In someembodiments, a linker comprises one or more fluorophores attached withinthe linker and one or more fluorophores attached via a spacer. Forexample, in some embodiments, a brightly labeled reactant describedherein comprises one or more (e.g., one, two, three, four, five, six, ormore than six) fluorophores attached within a linker and without aspacer, and one or more (e.g., one, two, three, four, five, six, or morethan six) fluorophores attached to the linker via a spacer.

Table 5. Examples of fluorophores for attachment within a linker

TABLE 5 Examples of fluorophores for attachment within a linker

For a set of luminescently labeled molecules (e.g., luminescentlylabeled nucleotides), the properties of a luminescently labeled FRETpair may allow for selection of a plurality of distinguishable molecules(e.g., nucleotides). In some embodiments, the second chromophore of aFRET pair has a luminescent lifetime distinct from a plurality of otherluminescently labeled molecules. In some embodiments, the secondchromophore of a FRET pair has a luminescent intensity distinct from aplurality of other luminescently labeled molecules. In some embodiments,the second chromophore of a FRET pair has a luminescent lifetime andluminescent intensity distinct from a plurality of other luminescentlylabeled molecules. In some embodiments, the second chromophore of a FRETpair emits photons in a spectral range distinct from a plurality ofother luminescently labeled molecules. In some embodiments, the firstchromophore of a FRET pair has a luminescent lifetime distinct from aplurality of luminescently labeled molecules. In certain embodiments,the FRET pair may absorb excitation energy in a spectral range distinctfrom a plurality of other luminescently labeled molecules. In certainembodiments, the FRET pair may absorb excitation energy in the samespectral range as one or more of a plurality of other luminescentlylabeled molecules.

For sequencing reactions, certain combinations of brightly labeledreactants may be preferred. In some embodiments, at least one of thebrightly labeled reactants comprises a cyanine dye, or analog thereof.In some embodiments, at least one of the brightly labeled reactantscomprises a rhodamine dye, or analog thereof. In some embodiments, atleast two of the brightly labeled reactants each comprises a cyaninedye, or analog thereof. In some embodiments, at least two of thebrightly labeled reactants each comprises a rhodamine dye, or analogthereof. In some embodiments, at least three of the brightly labeledreactants each comprises a cyanine dye, or analog thereof. In someembodiments, at least three of the brightly labeled reactants eachcomprises a rhodamine dye, or analog thereof. In some embodiments, atleast four of the brightly labeled reactants each comprises a cyaninedye, or analog thereof. In some embodiments, at least four of thebrightly labeled reactants each comprises a rhodamine dye, or analogthereof. In some embodiments, three of the brightly labeled reactantseach comprises a cyanine dye, or analog thereof, and a fourth brightlylabeled reactant comprises a rhodamine dye, or analog thereof. In someembodiments, two of the brightly labeled reactants each comprises acyanine dye, or analog thereof, and a third, and optionally a fourth,brightly labeled reactant comprises a rhodamine dye, or analog thereof.In some embodiments, three of the brightly labeled reactants eachcomprises a rhodamine dye, or analog thereof, and a third, andoptionally a fourth, brightly labeled reactant comprises a cyanine dye,or analog thereof.

As described herein, a luminescent label is a molecule that absorbs oneor more photons and may subsequently emit one or more photons after oneor more time durations. The luminescence of the molecule is described byseveral parameters, including but not limited to luminescent lifetime,absorption spectra, emission spectra, luminescent quantum yield, andluminescent intensity. The terms absorption and excitation are usedinterchangeably throughout the application. In some embodiments, theterms luminescence and emission are used interchangeably. A typicalluminescent molecule may absorb, or undergo excitation by, light atmultiple wavelengths. Excitation at certain wavelengths or withincertain spectral ranges may relax by a luminescent emission event, whileexcitation at certain other wavelengths or spectral ranges may not relaxby a luminescent emission event. In some embodiments, a luminescentmolecule is only suitably excited for luminescence at a singlewavelength or within a single spectral range. In some embodiments, aluminescent molecule is suitably excited for luminescence at two or morewavelengths or within two or more spectral ranges. In some embodiments,a molecule is identified by measuring the wavelength of the excitationphoton or the absorption spectrum.

The emitted photon from a luminescent emission event will emit at awavelength within a spectral range of possible wavelengths. Typicallythe emitted photon has a longer wavelength (e.g., has less energy or isred-shifted) compared to the wavelength of the excitation photon. Incertain embodiments, a molecule is identified by measuring thewavelength of an emitted photon. In certain embodiments, a molecule isidentified by measuring the wavelength of a plurality of emitted photon.In certain embodiments, a molecule is identified by measuring theemission spectrum.

Luminescent lifetime refers to the time duration between an excitationevent and an emission event. In some embodiments, luminescent lifetimeis expressed as the constant in an equation of exponential decay. Insome embodiments, wherein there are one or more pulse events deliveringexcitation energy, the time duration is the time between the pulse andthe subsequent emission event.

Determination of a luminescent lifetime of a molecule can be performedusing any suitable method (e.g., by measuring the lifetime using asuitable technique or by determining time-dependent characteristics ofemission). In some embodiments, determining the luminescent lifetime ofa molecule comprises determining the lifetime relative to one or moremolecules (e.g., different luminescently labeled nucleosidepolyphosphates in a sequencing reaction). In some embodiments,determining the luminescent lifetime of a molecule comprises determiningthe lifetime relative to a reference. In some embodiments, determiningthe luminescent lifetime of a molecule comprises measuring the lifetime(e.g., fluorescence lifetime). In some embodiments, determining theluminescent lifetime of a molecule comprises determining one or moretemporal characteristics that are indicative of lifetime. In someembodiments, the luminescent lifetime of a molecule can be determinedbased on a distribution of a plurality of emission events (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 60, 70, 80, 90, 100, or more emission events) occurring across oneor more time-gated windows relative to an excitation pulse. For example,a luminescent lifetime of a single molecule can be distinguished from aplurality of molecules having different luminescent lifetimes based onthe distribution of photon arrival times measured with respect to anexcitation pulse.

It should be appreciated that a luminescent lifetime of a singlemolecule is indicative of the timing of photons emitted after the singlemolecule reaches an excited state and the single molecule can bedistinguished by information indicative of the timing of the photons.Some embodiments may include distinguishing a molecule from a pluralityof molecules based on the molecule's luminescent lifetime by measuringtimes associated with photons emitted by the molecule. The distributionof times may provide an indication of the luminescent lifetime which maybe determined from the distribution. In some embodiments, the singlemolecule is distinguishable from the plurality of molecules based on thedistribution of times, such as by comparing the distribution of times toa reference distribution corresponding to a known molecule. In someembodiments, a value for the luminescent lifetime is determined from thedistribution of times.

Luminescent quantum yield refers to the fraction of excitation events ata given wavelength or within a given spectral range that lead to anemission event, and is typically less than 1. In some embodiments, theluminescent quantum yield of a molecule described herein is between 0and about 0.001, between about 0.001 and about 0.01, between about 0.01and about 0.1, between about 0.1 and about 0.5, between about 0.5 and0.9, or between about 0.9 and 1. In some embodiments, a molecule isidentified by determining or estimating the luminescent quantum yield.

As used herein for single molecules, luminescent intensity refers to thenumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy. Insome embodiments, the luminescent intensity refers to the detectednumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy, andare detected by a particular sensor or set of sensors.

The luminescent lifetime, luminescent quantum yield, and luminescentintensity may each vary for a given molecule under different conditions.In some embodiments, a single molecule will have a different observedluminescent lifetime, luminescent quantum yield, or luminescentintensity than for an ensemble of the molecules. In some embodiments, amolecule confined in a sample well will have a different observedluminescent lifetime, luminescent quantum yield, or luminescentintensity than for molecules not confined in a sample well. In someembodiments, a luminescent label or luminescent molecule attached toanother molecule will have a different luminescent lifetime, luminescentquantum yield, or luminescent intensity than the luminescent label orluminescent molecule not attached to another molecule. In someembodiments, a molecule interacting with a macromolecular complex willhave different luminescent lifetime, luminescent quantum yield, orluminescent intensity than a molecule not interacting with amacromolecular complex.

In certain embodiments, a luminescent molecule described in theapplication absorbs one photon and emits one photon after a timeduration. In some embodiments, the luminescent lifetime of a moleculecan be determined or estimated by measuring the time duration. In someembodiments, the luminescent lifetime of a molecule can be determined orestimated by measuring a plurality of time durations for multiple pulseevents and emission events. In some embodiments, the luminescentlifetime of a molecule can be differentiated amongst the luminescentlifetimes of a plurality of types of molecules by measuring the timeduration. In some embodiments, the luminescent lifetime of a moleculecan be differentiated amongst the luminescent lifetimes of a pluralityof types of molecules by measuring a plurality of time durations formultiple pulse events and emission events. In certain embodiments, amolecule is identified or differentiated amongst a plurality of types ofmolecules by determining or estimating the luminescent lifetime of themolecule. In certain embodiments, a molecule is identified ordifferentiated amongst a plurality of types of molecules bydifferentiating the luminescent lifetime of the molecule amongst aplurality of the luminescent lifetimes of a plurality of types ofmolecules.

In certain embodiments, the luminescent emission event is afluorescence. In certain embodiments, the luminescent emission event isa phosphorescence. As used herein, the term luminescence encompasses allluminescent events including both fluorescence and phosphorescence.

Sequencing

Some aspects of the application are useful for sequencing biologicalpolymers, such as nucleic acids and proteins. In some aspects,compositions and techniques described in the application can be used toidentify a series of nucleotide or amino acid monomers that areincorporated into a nucleic acid or protein (e.g., by detecting atime-course of incorporation of a series of labeled nucleotide or aminoacid monomers). In some embodiments, compositions and techniquesdescribed in the application can be used to identify a series ofnucleotides that are incorporated into a template-dependent nucleic acidsequencing reaction product synthesized by a polymerase enzyme.

Upon base pairing between a nucleobase of a target nucleic acid and thecomplementary nucleoside polyphosphate (e.g., dNTP), the polymeraseincorporates the dNTP into the newly synthesized nucleic acid strand byforming a phosphodiester bond between the 3′ hydroxyl end of the newlysynthesized strand and the alpha phosphate of the dNTP. In examples inwhich the luminescent label conjugated to the dNTP (e.g., through alinker of the application) is a fluorophore, its presence is signaled byexcitation and a pulse of emission is detected during and/or after thestep of incorporation. For detection labels (e.g., luminescent labels)that are conjugated, through a linker of the application, to theterminal (gamma) phosphate of the dNTP, incorporation of the dNTP intothe newly synthesized strand results in release of the beta and gammaphosphates and the linker comprising the detection label, which is freeto diffuse in the sample well, resulting in a decrease in emissiondetected from the fluorophore.

In certain embodiments, the template-dependent nucleic acid sequencingreaction product is synthesized in a sequencing reaction carried out bynaturally occurring nucleic acid polymerases. In some embodiments, thepolymerase is a mutant or modified variant of a naturally occurringpolymerase. In some embodiments, the template-dependent nucleic acidsequencing product will comprise one or more nucleotide segmentscomplementary to the template nucleic acid strand. In one aspect, theapplication provides a method of determining the sequence of a template(or target) nucleic acid strand by determining the sequence of itscomplementary nucleic acid strand.

The term “polymerase,” as used herein, generally refers to any enzyme(or polymerizing enzyme) capable of catalyzing a polymerizationreaction. Examples of polymerases include, without limitation, a nucleicacid polymerase, a transcriptase or a ligase. A polymerase can be apolymerization enzyme. Embodiments directed towards single moleculenucleic acid extension (e.g., for nucleic acid sequencing) may use anypolymerase that is capable of synthesizing a nucleic acid complementaryto a target nucleic acid molecule. In some embodiments, a polymerase maybe a DNA polymerase, an RNA polymerase, a reverse transcriptase, and/ora mutant or altered form of one or more thereof.

Examples of polymerases include, but are not limited to, a DNApolymerase, an RNA polymerase, a thermostable polymerase, a wild-typepolymerase, a modified polymerase, E. coli DNA polymerase I, T7 DNApolymerase, bacteriophage T4 DNA polymerase, ϕ29 (phi29) DNA polymerase,Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwopolymerase, Vent® polymerase, Deep Vent® polymerase, ExTaq™ polymerase,LA Taq™ polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tnepolymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfipolymerase, Platinum® Taq polymerases, Tbr polymerase, Tfl polymerase,Tth polymerase, PfuTurbo polymerase, Pyrobest™ polymerase, Pwopolymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenowfragment, polymerase with 3′ to 5′ exonuclease activity, and variants,modified products and derivatives thereof. In some embodiments, thepolymerase is a single subunit polymerase. Non-limiting examples of DNApolymerases and their properties are described in detail in, among otherplaces, DNA Replication 2nd edition, Kornberg and Baker, W. H. Freeman,New York, N.Y. (1991).

In some embodiments, the polymerase is a polymerase with highprocessivity. However, in some embodiments, the polymerase is apolymerase with reduced processivity. Polymerase processivity generallyrefers to the capability of a polymerase to consecutively incorporatedNTPs into a nucleic acid template without releasing the nucleic acidtemplate. In some embodiments, the polymerase is a polymerase with low5′-3′ exonuclease activity and/or 3′-5′ exonuclease. In someembodiments, the polymerase is modified (e.g., by amino acidsubstitution) to have reduced 5′-3′ exonuclease activity and/or 3′-5′activity relative to a corresponding wild-type polymerase. Furthernon-limiting examples of DNA polymerases include 9° Nm™ DNA polymerase(New England Biolabs), and a P680G mutant of the Klenow exo-polymerase(Tuske et al. (2000) JBC 275(31):23759-23768). In some embodiments, apolymerase having reduced processivity provides increased accuracy forsequencing templates containing one or more stretches of nucleotiderepeats (e.g., two or more sequential bases of the same type). In someembodiments, the polymerase is a polymerase that has a higher affinityfor a labeled nucleotide than for a non-labeled nucleic acid.

In another aspect, the application provides methods of sequencing targetnucleic acids by sequencing a plurality of nucleic acid fragments,wherein the target nucleic acid comprises the fragments. In certainembodiments, the method comprises combining a plurality of fragmentsequences to provide a sequence or partial sequence for the parenttarget nucleic acid. In some embodiments, the step of combining isperformed by computer hardware and software. The methods describedherein may allow for a set of related target nucleic acids, such as anentire chromosome or genome to be sequenced.

During sequencing, a polymerizing enzyme may couple (e.g., attach) to apriming location of a target nucleic acid molecule. The priming locationcan be a primer that is complementary to a portion of the target nucleicacid molecule. As an alternative, the priming location is a gap or nickthat is provided within a double stranded segment of the target nucleicacid molecule. A gap or nick can be from 0 to at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, or 40 nucleotides in length. A nick can provide abreak in one strand of a double stranded sequence, which can provide apriming location for a polymerizing enzyme, such as, for example, astrand displacing polymerase enzyme.

In some cases, a sequencing primer can be annealed to a target nucleicacid molecule that may or may not be immobilized to a solid support. Asolid support can comprise, for example, a sample well (e.g., ananoaperture, a reaction chamber) on a chip used for nucleic acidsequencing. In some embodiments, a sequencing primer may be immobilizedto a solid support and hybridization of the target nucleic acid moleculealso immobilizes the target nucleic acid molecule to the solid support.In some embodiments, a polymerase is immobilized to a solid support andsoluble primer and target nucleic acid are contacted to the polymerase.However, in some embodiments, a complex comprising a polymerase, atarget nucleic acid and a primer is formed in solution and the complexis immobilized to a solid support (e.g., via immobilization of thepolymerase, primer, and/or target nucleic acid). In some embodiments,none of the components in a sample well (e.g., a nanoaperture, areaction chamber) are immobilized to a solid support. For example, insome embodiments, a complex comprising a polymerase, a target nucleicacid, and a primer is formed in solution and the complex is notimmobilized to a solid support.

Under appropriate conditions, a polymerase enzyme that is contacted toan annealed primer/target nucleic acid can add or incorporate one ormore nucleotides onto the primer, and nucleotides can be added to theprimer in a 5′ to 3′, template-dependent fashion. Such incorporation ofnucleotides onto a primer (e.g., via the action of a polymerase) cangenerally be referred to as a primer extension reaction. Each nucleotidecan be associated with a detectable label that can be detected andidentified (e.g., based on its luminescent lifetime and/or othercharacteristics) during the nucleic acid extension reaction and used todetermine each nucleotide incorporated into the extended primer and,thus, a sequence of the newly synthesized nucleic acid molecule. Viasequence complementarity of the newly synthesized nucleic acid molecule,the sequence of the target nucleic acid molecule can also be determined.In some cases, annealing of a sequencing primer to a target nucleic acidmolecule and incorporation of nucleotides to the sequencing primer canoccur at similar reaction conditions (e.g., the same or similar reactiontemperature) or at differing reaction conditions (e.g., differentreaction temperatures). In some embodiments, sequencing by synthesismethods can include the presence of a population of target nucleic acidmolecules (e.g., copies of a target nucleic acid) and/or a step ofamplification of the target nucleic acid to achieve a population oftarget nucleic acids. However, in some embodiments sequencing bysynthesis is used to determine the sequence of a single molecule in eachreaction that is being evaluated (and nucleic acid amplification is notrequired to prepare the target template for sequencing). In someembodiments, a plurality of single molecule sequencing reactions areperformed in parallel (e.g., on a single chip) according to aspects ofthe present application. For example, in some embodiments, a pluralityof single molecule sequencing reactions are each performed in separatereaction chambers (e.g., nanoapertures, sample wells) on a single chip.

Embodiments are capable of sequencing single nucleic acid molecules withhigh accuracy and long read lengths, such as an accuracy of at leastabout 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, or 99.9999%, and/or read lengths greater than or equalto about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or100,000 bp. In some embodiments, the target nucleic acid molecule usedin single molecule sequencing is a single stranded target nucleic acid(e.g., deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid(RNA), RNA derivatives) template that is added or immobilized to asample well (e.g., nanoaperture) containing at least one additionalcomponent of a sequencing reaction (e.g., a polymerase such as, a DNApolymerase, a sequencing primer) immobilized or attached to a solidsupport such as the bottom or side walls of the sample well. The targetnucleic acid molecule or the polymerase can be attached to a samplewall, such as at the bottom or side walls of the sample well directly orthrough a linker. The sample well (e.g., nanoaperture) also can containany other reagents needed for nucleic acid synthesis via a primerextension reaction, such as, for example suitable buffers, co-factors,enzymes (e.g., a polymerase) and deoxyribonucleoside polyphosphates,such as, e.g., deoxyribonucleoside triphosphates, includingdeoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP),and deoxythymidine triphosphate (dTTP) dNTPs, that include luminescentlabels, such as fluorophores, which can be connected to the dNTPsthrough a linker of the application. In some embodiments, each class ofdNTPs (e.g., adenine-containing dNTPs (e.g., dATP), cytosine-containingdNTPs (e.g., dCTP), guanine-containing dNTPs (e.g., dGTP),uracil-containing dNTPs (e.g., dUTPs) and thymine-containing dNTPs(e.g., dTTP)) is conjugated (e.g., through a linker of the application)to a distinct luminescent label such that detection of light emittedfrom the label indicates the identity of the dNTP that was incorporatedinto the newly synthesized nucleic acid. A “distinct luminescent label”can, in some embodiments, refer to one dNTP that comprises a differentluminescent label (e.g., a different fluorophore) than another dNTP. Insome embodiments, a distinct luminescent label refers to one dNTP thatcomprises a different number of the same or similar luminescent label asanother dNTP. In some embodiments, a distinct luminescent label refersto one dNTP that comprises one or more luminescent properties that aredetectably different from another dNTP. Emitted light from theluminescent label can be detected and attributed to its appropriateluminescent label (and, thus, associated dNTP) via any suitable deviceand/or method. The luminescent label may be conjugated (e.g., through alinker of the application) to the dNTP at any position such that thepresence of the luminescent label does not inhibit the incorporation ofthe dNTP into the newly synthesized nucleic acid strand or the activityof the polymerase. In some embodiments, the luminescent label isconjugated (e.g., through a linker of the application) to the terminalphosphate (e.g., the gamma phosphate) of the dNTP.

In some embodiments, the single-stranded target nucleic acid templatecan be contacted with a sequencing primer, dNTPs, polymerase and otherreagents necessary for nucleic acid synthesis. In some embodiments, allappropriate dNTPs can be contacted with the single-stranded targetnucleic acid template simultaneously (e.g., all dNTPs are simultaneouslypresent) such that incorporation of dNTPs can occur continuously. Inother embodiments, the dNTPs can be contacted with the single-strandedtarget nucleic acid template sequentially, where the single-strandedtarget nucleic acid template is contacted with each appropriate dNTPseparately, with washing steps in between contact of the single-strandedtarget nucleic acid template with differing dNTPs. Such a cycle ofcontacting the single-stranded target nucleic acid template with eachdNTP separately followed by washing can be repeated for each successivebase position of the single-stranded target nucleic acid template to beidentified.

In some embodiments, the sequencing primer anneals to thesingle-stranded target nucleic acid template and the polymeraseconsecutively incorporates the dNTPs (or other nucleoside polyphosphate)to the primer based on the single-stranded target nucleic acid template.The unique luminescent label associated with each incorporated dNTP canbe excited with the appropriate excitation light during or afterincorporation of the dNTP to the primer and its emission can besubsequently detected, using, any suitable device(s) and/or method(s).Detection of a particular emission of light (e.g., having a particularemission lifetime, intensity, spectrum and/or combination thereof) canbe attributed to a particular dNTP incorporated. The sequence obtainedfrom the collection of detected luminescent labels can then be used todetermine the sequence of the single-stranded target nucleic acidtemplate via sequence complementarity.

In some embodiments, the present disclosure provides methods andcompositions that may be advantageously utilized in the technologiesdescribed in co-pending U.S. patent application Ser. Nos. 14/543,865,14/543,867, 14/543,888, 14/821,656, 14/821,686, 14/821,688, 15/161,067,15/161,088, 15/161,125, 15/255,245, 15/255,303, 15/255,624, 15/261,697,15/261,724, 15/600,979, 15/846,967, 15/847,001, 15/971,493, 62/289,019,62/296,546, 62/310,398, 62/339,790, 62/343,997, 62/344,123, and62/426,144, the contents of each of which are incorporated herein byreference.

Kits

In yet other aspects, the application provides kits for sequencing atemplate nucleic acid. In some embodiments, a kit comprises a pluralityof types of luminescently labeled nucleotides as described herein. Insome embodiments, each type of labeled nucleotide comprises two or moreluminescent labels attached to one or more nucleoside polyphosphates viaa linker according to the application. In some embodiments, theplurality of nucleotides are selected from the labeled nucleotidesdepicted in FIGS. 3A-3C, 3E-3G, 4A-4C, 5A-5B, 6A-6C, and 8-10 . Forexample, in some embodiments, the plurality of nucleotides are designedaccording to the structures shown in FIG. 3G. In some embodiments, theplurality of nucleotides are designed according to the structures shownin FIG. 3B (306), FIG. 3B (308), and FIG. 5A (502). In some embodiments,the kit further comprises a polymerizing enzyme (e.g., a DNA polymerase,as described elsewhere herein). In some embodiments, the kit furthercomprises a primer complementary to the template nucleic acid beingsequenced.

In some aspects, the application provides reaction mixtures comprisingone or more of the brightly labeled reactants described herein. In someembodiments, the reaction mixture comprises a mixture added to asequencing reaction. In some embodiments, the reaction mixture includesa polymerizing enzyme. In some embodiments, the polymerizing enzyme isconfigured to be immobilized to a solid support (e.g., the bottom of asample well, as described elsewhere herein). In some embodiments, thereaction mixture comprises a template nucleic acid to be sequenced. Insome embodiments, the reaction mixture comprises a primer complementaryto a portion of the template nucleic acid. In some embodiments, thereaction mixture comprises one or more components necessary to initiatea sequencing reaction (e.g., a divalent metal ion, such as magnesium oriron). In some embodiments, the reaction mixture comprises one or morecomponents necessary to stabilize a sequencing reaction (e.g., one ormore buffering agents, one or more reducing agents, etc.).

EXAMPLES Example 1: Dye Attachment Strategies with Nucleic Acid Linkers

Various next-generation sequencing technologies implement labeledreaction components. For example, dye-labeled nucleotides can be used tomake specific base calls during incorporation events based on thedetection or observation of unique luminescent properties correspondingto each base type. These properties, such as lifetime and intensity,must therefore be readily identifiable for each base among a set.Initial efforts into enhancing fluorescence intensity of labelednucleotides revealed that the brightness of a dye-labeled nucleotide wasincreased when a second dye molecule was added to the construct.However, fluorescence lifetime was noticeably decreased relative to thesingly-labeled variant. In developing improved labeled nucleotides,nucleic acids were investigated as core structures for linkingfluorescent dyes to nucleotides.

One potential source of altered fluorescence lifetime inmultiply-labeled nucleotides is the extent of interaction between dyemolecules of the same construct, which can result in a quenching effect.This possibility was explored further by generating the dye-labelednucleotides shown in FIG. 8 . Two dye molecules (DyLight® 530R2) wereconnected to a nucleoside polyphosphate via a nucleic acid linker. Anunlabeled oligonucleotide strand was hybridized with the labeledoligonucleotide strand to impart rigidity in the nucleic acid linker. Afirst construct 800 was made using C6-amino-T spacers for dye attachmentto the nucleic acid, and a second construct 802 was made usingglycolamine spacers for dye attachment.

Analysis of the first construct revealed a fluorescence lifetime ofapproximately 1.4 ns, while the second construct exhibited a lifetime ofapproximately 3.5 ns. The measured increase in lifetime with the secondconstruct was attributed to the use of relatively shorter spacers fordye attachment in comparison to the first construct. As shown in FIG. 8, the C6-amino-T spacers of the first construct are of greater lengththan the glycolamine spacers of the second construct. One possibleexplanation for the improved lifetime of the second construct is thatthe shortened spacer length decreased dye-dye interactions by limitingthe extent to which the ranges of movement of the attached dyesoverlapped.

Example 2: Increased Linker Rigidity Prolongs Fluorescence Lifetime

Following observations that spacer length can affect fluorescencelifetime, potentially due to spatial overlap of dyes, it was thoughtthat a symmetrical arrangement of dyes in a multiply-labeled moleculemight have similar effects. A Y-shaped nucleic acid linker was generatedand is shown in FIG. 9 . The initial construct had three oligonucleotidestrands covalently attached via the branched linker shown. Two strandswere each terminally attached to a dye molecule (Chromis 530N), whereasthe third strand was terminally attached to a nucleoside polyphosphate.The third strand was further hybridized with an unlabeled strand toimpart rigidity between the nucleoside polyphosphate and the labeledregion. A second version of this construct was generated byhybridization with oligonucleotide component 902, which hybridizes tothe first and second strands.

Analysis of the initial construct revealed a fluorescence lifetime ofapproximately 2.3 ns, while the construct having the additionaloligonucleotide component 902 exhibited a lifetime of approximately 4.2ns. One potential explanation for the measured increase in lifetime withthe latter construct is that the oligonucleotide component 902 impartedrigidity in the labeled region. The increased rigidity could conceivablypromote dye separation by constraining each dye to a more limited rangeof movement.

Example 3: Effects of Dye Spacer Length in Constrained LinkerConfigurations

Geometrically constrained linker configurations were further developedby generating the tris-dye labeled construct shown in FIG. 10 . Asshown, the nucleic acid linker portion included three mainoligonucleotide components. The first component included fouroligonucleotide strands covalently attached via the four-way branchedlinker shown. Three of these strands were each terminally attached to adye molecule (ATTO™ Rho6G(N-(9-(2-((3-carboxypropyl)(methyl)carbamoyl)phenyl)-6-(ethylamino)-2,7-dimethyl-3H-xanthen-3-ylidene)-N-methylmethanaminium)),whereas the fourth strand was hybridized with a second oligonucleotidecomponent. The second oligonucleotide component was terminally attachedto two nucleoside polyphosphates via the branched linker shown. Thethird oligonucleotide component, which included three oligonucleotidestrands covalently attached via the branched linker shown, washybridized with the three dye-labeled strands of the first component toimpart rigidity in the labeled region. Two separate tris-dye constructshaving differing spacer lengths were generated according to the boxedarea shown in FIG. 10 .

The first tris-dye labeled nucleotide construct having a longer spacer(see FIG. 10 , boxed area, top) showed a tripling in fluorescenceintensity relative to a one-dye labeled nucleotide construct.Additionally, it was noted that lifetime was slightly reduced whencompared to a two-dye, one nucleotide construct having the same dyemolecule (not shown). Measurements obtained for the second tris-dyelabeled nucleotide construct having a shorter spacer (see FIG. 10 ,boxed area, bottom) showed a slight increase in fluorescence lifetimerelative to the first tris-dye labeled construct.

Earlier tris-dye labeled nucleotides produced multiple lifetimes duringsequencing reactions, which was thought to be the result of two dyesinteracting to produce a first lifetime while the non-interacting dyeproduced a second lifetime. Importantly, only a single lifetime wasobserved for either tris-dye labeled molecule shown in FIG. 10 duringsequencing reactions.

EQUIVALENTS AND SCOPE

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents, and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03. It should be appreciatedthat embodiments described in this document using an open-endedtransitional phrase (e.g., “comprising”) are also contemplated, inalternative embodiments, as “consisting of” and “consisting essentiallyof” the feature described by the open-ended transitional phrase. Forexample, if the disclosure describes “a composition comprising A and B,”the disclosure also contemplates the alternative embodiments “acomposition consisting of A and B” and “a composition consistingessentially of A and B.”

The invention claimed is:
 1. A labeled molecule comprising a reactantconnected to two or more luminescent labels via a nucleic acid linker,wherein each luminescent label is at least 5 angstroms separated fromany other luminescent label, wherein (i) at least one luminescent labelis integrated within the nucleic acid linker, and wherein the at leastone luminescent label that is integrated within the nucleic acid linkeris a cyanine-based fluorophore; and/or (ii) wherein at least oneluminescent label is attached to the nucleic acid linker via aglycolamine spacer molecule, and wherein the labeled nucleotidecomprises the structure:

where

is the at least one luminescent label that is attached to the nucleicacid linker via the glycolamine spacer molecule.
 2. The labeled moleculeof claim 1, wherein the cyanine-based fluorophore has the chemicalstructure:


3. The labeled molecule of claim 1, wherein the at least one luminescentlabel attached to the nucleic acid linker via a glycolamine spacermolecule comprises a rhodamine dye, a BODIPY dye, or a cyanine dye.
 4. Alabeled nucleotide comprising a nucleoside polyphosphate connected totwo or more luminescent labels via a nucleic acid linker, wherein thenucleic acid linker is connected to the terminal phosphate of thenucleoside polyphosphate, wherein each luminescent label is at least 5angstroms separated from any other luminescent label, wherein (i) atleast one luminescent label is integrated into the nucleic acid linker,and wherein the at least one luminescent label that is integrated withinthe nucleic acid linker is a cyanine-based fluorophore; and/or (ii)wherein at least one luminescent label is attached to the nucleic acidlinker via a glycolamine spacer molecule, and wherein the labelednucleotide comprises the structure:

where

is the at least one luminescent label that is attached to the nucleicacid linker via the glycolamine spacer molecule.
 5. The labelednucleotide of claim 4, wherein each luminescent label comprises a centerof mass that is at least 5 angstroms separated from the center of massof any other luminescent label.
 6. The labeled nucleotide of claim 4,wherein the cyanine-based fluorophore has the chemical structure:


7. The labeled nucleotide of claim 4, wherein the at least oneluminescent label attached to the nucleic acid linker via a glycolaminespacer molecule comprises a rhodamine dye, a BODIPY dye, or a cyaninedye.
 8. The labeled nucleotide of claim 4, wherein the nucleic acidlinker is an oligomer that comprises at least 10 monomeric units.
 9. Thelabeled nucleotide of claim 8, wherein the oligomer comprises fewer than150, fewer than 100, or fewer than 50 monomeric units.
 10. The labelednucleotide of claim 8, wherein at least one luminescent label isattached to the nucleic acid linker at an attachment site that is atleast 5 monomeric units separated from any other attachment site. 11.The labeled nucleotide of claim 4, wherein the nucleic acid linkercomprises a first oligonucleotide strand attached to the two or moreluminescent labels at two or more attachment sites on the firstoligonucleotide strand, and wherein each luminescent label comprises asteric volume having a center point that is at least 5 angstromsseparated from that of any other luminescent label.
 12. The labelednucleotide of claim 11, further comprising a second oligonucleotidestrand hybridized with the first oligonucleotide strand, wherein thefirst oligonucleotide strand is attached to the nucleosidepolyphosphate.
 13. The labeled nucleotide of claim 11, furthercomprising a second oligonucleotide strand hybridized with the firstoligonucleotide strand, wherein the second oligonucleotide strand isattached to the nucleoside polyphosphate.
 14. The labeled nucleotide ofclaim 11, wherein the two or more attachment sites are separated fromone another by at least 5 and fewer than 50 bases on the firstoligonucleotide strand.
 15. The labeled nucleotide of claim 11, whereineach attachment site is at least 2 bases separated from a guanine or acytosine on the first oligonucleotide strand.
 16. The labeled nucleotideof claim 11, wherein at least one attachment site occurs at an abasicsite on the first oligonucleotide strand.
 17. The labeled nucleotide ofclaim 11, wherein the first oligonucleotide strand forms one or morestem-loops.
 18. The labeled nucleotide of claim 17, wherein a loopregion of each stem-loop comprises an attachment site of the two or moreattachment sites.